Derivatives of 1,2-dihydro-7-hydroxyquinolines containing fused rings

ABSTRACT

The present invention describes novel dyes, including coumarins, rhodamines, and rhodols that incorporate additional fused aromatic rings. The dyes of the invention absorb at a longer wavelength than structurally similar dyes that do not possess the fused aromatic rings. Many of the dyes of the invention are useful fluorescent dyes. The invention includes chemically reactive dyes, dye-conjugates, and the use of such dyes in staining samples and detecting ligands or other analytes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 11/560,579, filedNov. 16, 2006, now U.S. Pat. No. 7,816,519, which is a continuation ofU.S. Ser. No. 10/713,670, now U.S. Pat. No. 7,169,922, filed on Nov. 13,2003, which is a continuation-in-part of U.S. Ser. No. 09/922,333, nowU.S. Pat. No. 6,716,979, filed Aug. 4, 2001, which claims priority toU.S. Ser. No. 60/223,086, filed Aug 4, 2000, the disclosures of whichare herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to colored and fluorescent dyes, includingreactive dye derivatives, and dye-conjugates; and to their use instaining samples and detecting ligands or other analytes.

BACKGROUND OF THE INVENTION

Fluorescent dyes are known to be particularly suitable for applicationsin which a highly sensitive detection reagent is desirable. Fluorescentdyes are used to impart both visible color and fluorescence to othermaterials. As researchers increasingly utilize fluorescent probes asresearch tools, the ability to select the wavelength of fluorescencebecomes more important, particularly as more multiple-color applicationsare developed.

A variety of fluorescent dyes have been previously and extensivelydescribed, including coumarins, fluoresceins, rhodamines, rhodols,oxazines, carbocyanines, and derivatives thereof. The selection ofcertain substituents has been shown to be useful in adjusting thespectral properties of such dyes but there have remained regions of thevisible spectrum where suitable fluorescent dyes either did not exist,or did not possess particularly favorable properties.

The dyes of the invention incorporate additional fused aromatic orheteroaromatic rings, and exhibit a shift of fluorescence emission tolonger wavelength that is typically greater than 20 nm, relative tootherwise structurally similar dyes known in the art. This bathochromicspectral shift yields dyes that are particularly useful for excitationin the wavelength ranges between 400 nm and 600 nm and in particular atgreater than 630 nm. Of particular importance are the dyes of theinvention that exhibit absorbance maxima between 530 nm and 650 nm, asthey match the principal emission lines of the mercury arc lamp (546nm), frequency-doubled Nd-Yag laser (532 nm), Kr-ion laser (568 nm, and647 nm) and HeNe laser (543 nm, 594 nm, and 633 nm).

Fluorescent dyes of the invention with longer wavelength absorption andemission are particularly useful in conjunction with materials ofbiological origin such as blood, urine, fecal matter, cells and tissues,because background or inherent fluorescence or absorption is less likelyto interfere with dye detection. Furthermore, infrared dyes of theinvention have enhanced utility in biological systems that aretransparent at infrared wavelengths. The long wavelength dyes of theinvention also have advantages in use as laser dyes, or in electronicsas optical memory elements using relatively low cost illuminationsources such as laser diodes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: The excitation and emission spectra of a streptavidin conjugateof Compound 42.

FIG. 2: A comparison of the rate of photobleaching between Compound 42and the fluorescent dye CY-5 in phosphate-buffered saline, as describedin Example 54.

FIG. 3: The effect of pH on the fluorescence emission of Compound 57, asdescribed in Example 44.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

The present invention describes derivatives of1,2-dihydro-7-hydroxyquinoline useful for the preparation of a varietyof fluorescent dyes and dye derivatives. In the derivatives describedherein, the 3,4-double bond of the dihydroquinoline is fused to anaromatic or heteroaromatic ring that is in turn optionally fused to oneor more additional aromatic or heteroaromatic rings. The invention alsoincludes dyes that are prepared from the novel synthetic precursors,including, but not limited to, coumarin compounds, rhodamine compounds,rhodol compounds, triarylmethane compounds, phenoxazine compounds, andtheir benzo and annelated derivatives. The dyes of the inventionoptionally possess a reactive group useful for preparing fluorescentconjugates, which conjugates and methods for their preparation and useare described herein.

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositionsor process steps, as such may vary. It must be noted that, as used inthis specification and the appended claims, the singular form “a”, “an”and “the” includes plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a fluorescent dye” includesa plurality of dyes and reference to “a compound” includes a pluralityof compounds and the like.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention is related. The following terms aredefined for purposes of the invention as described herein.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are encompassed within thescope of the present invention.

The compounds of the invention may be prepared as a single isomer (e.g.,enantiomer, cis-trans, positional, diastereomer) or as a mixture ofisomers. In a preferred embodiment, the compounds are prepared assubstantially a single isomer. Methods of preparing substantiallyisomerically pure compounds are known in the art. For example,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Alternatively, the final product or intermediates along the syntheticroute can be resolved into a single stereoisomer. Techniques forinverting or leaving unchanged a particular stereocenter, and those forresolving mixtures of stereoisomers are well known in the art and it iswell within the ability of one of skill in the art to choose andappropriate method for a particular situation. See, generally, Furnisset al. (eds.), VOGEL'S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY5^(TH) ED., Longman Scientific and Technical Ltd., Essex, 1991, pp.809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

Although typically not shown for the sake of clarity, any overallpositive or negative charges possessed by any of the compounds of theinvention are balanced by a necessary counterion or counterions. Wherethe compound of the invention is positively charged, the counterion istypically selected from, but not limited to, chloride, bromide, iodide,sulfate, alkanesulfonate, arylsulfonate, phosphate, perchlorate,tetrafluoroborate, tetraarylborate, nitrate, hexafluorophosphate, andanions of aromatic or aliphatic carboxylic acids. Where the compound ofthe invention is negatively charged, the counterion is typicallyselected from, but not limited to, alkali metal ions, alkaline earthmetal ions, transition metal ions, ammonium or substituted ammoniumions. Preferably, any necessary counterion is biologically compatible,is not toxic as used, and does not have a substantially deleteriouseffect on biomolecules. Counterions are readily changed by methods wellknown in the art, such as ion-exchange chromatography, or selectiveprecipitation.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents, which would result from writing thestructure from right to left, e.g., —CH₂O— is intended to also recite—OCH₂—.

The term “acyl” or “alkanoyl” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and an acyl radical onat least one terminus of the alkane radical. The “acyl radical” is thegroup derived from a carboxylic acid by removing the —OH moietytherefrom.

The term “alkyl,” by itself or as part of another substituent means,unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include divalent(“alkylene”) and multivalent radicals, having the number of carbon atomsdesignated (i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include, but are not limited to, groups such asmethyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl,sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologsand isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, andthe like. An unsaturated alkyl group is one having one or more doublebonds or triple bonds. Examples of unsaturated alkyl groups include, butare not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and3-propynyl, 3-butynyl, and the higher homologs and isomers. The term“alkyl,” unless otherwise noted, is also meant to include thosederivatives of alkyl defined in more detail below, such as“heteroalkyl.” Alkyl groups that are limited to hydrocarbon groups aretermed “homoalkyl”.

Exemplary alkyl groups of use in the present invention contain betweenabout one and about twenty-five carbon atoms (e.g. methyl, ethyl and thelike). Straight, branched or cyclic hydrocarbon chains having eight orfewer carbon atoms will also be referred to herein as “lower alkyl”. Inaddition, the term “alkyl” as used herein further includes one or moresubstitutions at one or more carbon atoms of the hydrocarbon chainfragment.

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a straight or branched chain, or cycliccarbon-containing radical, or combinations thereof, consisting of thestated number of carbon atoms and at least one heteroatom selected fromthe group consisting of O, N, Si, P and S, and wherein the nitrogen,phosphorous and sulfur atoms are optionally oxidized, and the nitrogenheteroatom is optionally be quaternized, and the sulfur atoms areoptionally trivalent with alkyl or heteroalkyl substituents. Theheteroatom(s) O, N, P, S and Si may be placed at any interior positionof the heteroalkyl group or at the position at which the alkyl group isattached to the remainder of the molecule. Examples include, but are notlimited to, —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, and —CH═CH—N(CH₃)—CH₃. Up to two heteroatomsmay be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. Similarly, the term “heteroalkylene” by itself or aspart of another substituent means a divalent radical derived fromheteroalkyl, as exemplified, but not limited by, —CH₂—CH₂—S—CH₂—CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini (e.g., alkyleneoxy,alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Stillfurther, for alkylene and heteroalkylene linking groups, no orientationof the linking group is implied by the direction in which the formula ofthe linking group is written. For example, the formula —C(O)₂R′—represents both —C(O)₂R′— and —R′C(O)₂—.

The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or incombination with other terms, represent, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl”, respectively. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic moiety that can be a single ring or multiple rings (preferablyfrom 1 to 4 rings), which are fused together or linked covalently.Specific examples of aryl substituents include, but are not limited to,substituted or unsubstituted derivatives of phenyl, biphenyl, o-, m-, orp-terphenyl, 1-naphthyl, 2-naphthyl, 1-, 2-, or 9-anthryl, 1-, 2-, 3-,4-, or 9-phenanthrenyl and 1-, 2- or 4-pyrenyl. Preferred arylsubstituents are phenyl, substituted phenyl, naphthyl or substitutednaphthyl.

The term “heteroaryl” as used herein refers to an aryl group as definedabove in which one or more carbon atoms have been replaced by anon-carbon atom, especially nitrogen, oxygen, or sulfur. For example,but not as a limitation, such groups include furyl, tetrahydrofuryl,pyrrolyl, pyrrolidinyl, thienyl, tetrahydrothienyl, oxazolyl,isoxazolyl, triazolyl, thiazolyl, isothiazolyl, pyrazolyl,pyrazolidinyl, oxadiazolyl, thiadiazolyl, imidazolyl, imidazolinyl,pyridyl, pyridaziyl, triazinyl, piperidinyl, morpholinyl,thiomorpholinyl, pyrazinyl, piperainyl, pyrimidinyl, naphthyridinyl,benzofuranyl, benzothienyl, indolyl, indolinyl, indolizinyl, indazolyl,quinolizinyl, qunolinyl, isoquinolinyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, pteridinyl, quinuclidinyl, carbazolyl,acridinyl, phenazinyl, phenothizinyl, phenoxazinyl, purinyl,benzimidazolyl and benzthiazolyl and their aromatic ring-fused analogs.Many fluorophores are comprised of heteroaryl groups and include,without limitations, xanthenes, oxazines, benzazolium derivatives(including cyanines and carbocyanines), borapolyazaindacenes,benzofurans, indoles and quinazolones.

Where a ring substituent is a heteroaryl substituent, it is defined as a5- or 6-membered heteroaromatic ring that is optionally fused to anadditional six-membered aromatic ring(s), or is fused to one 5- or6-membered heteroaromatic ring. The heteroaromatic rings contain atleast 1 and as many as 3 heteroatoms that are selected from the groupconsisting of O, N or S in any combination. The heteroaryl substituentis bound by a single bond, and is optionally substituted as definedbelow.

Specific examples of heteroaryl moieties include, but are not limitedto, substituted or unsubstituted derivatives of 2- or 3-furanyl; 2- or3-thienyl; N-, 2- or 3-pyrrolyl; 2- or 3-benzofuranyl; 2- or3-benzothienyl; N-, 2- or 3-indolyl; 2-, 3- or 4-pyridyl; 2-, 3- or4-quinolyl; 1-, 3-, or 4-isoquinolyl; 2-, 4-, or 5-(1,3-oxazolyl);2-benzoxazolyl; 2-, 4-, or 5-(1,3-thiazolyl); 2-benzothiazolyl; 3-, 4-,or 5-isoxazolyl; N-, 2-, or 4-imidazolyl; N-, or 2-benzimidazolyl; 1- or2-naphthofuranyl; 1- or 2-naphthothienyl; N-, 2- or 3-benzindolyl; 2-,3-, or 4-benzoquinolyl; 1-, 2-, 3-, or 4-acridinyl. Preferred heteroarylsubstituents include substituted or unsubstituted 4-pyridyl, 2-thienyl,2-pyrrolyl, 2-indolyl, 2-oxazolyl, 2-benzothiazolyl or 2-benzoxazolyl.

The above heterocyclic groups may further include one or moresubstituents at one or more carbon and/or non-carbon atoms of theheteroaryl group, e.g., alkyl; aryl; heterocycle; halogen; nitro; cyano;hydroxyl, alkoxyl or aryloxyl; thio or mercapto, alkyl- or arylthio;amino, alkyl-, aryl-, dialkyl-, diaryl-, or arylalkylamino;aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl,dialkylaminocarbonyl, diarylaminocarbonyl or arylalkylaminocarbonyl;carboxyl, or alkyl- or aryloxycarbonyl; aldehyde; aryl- oralkylcarbonyl; iminyl, or aryl- or alkyliminyl; sulfo; alkyl- orarylsulfonyl; hydroximinyl, or aryl- or alkoximinyl. In addition, two ormore alkyl substituents may be combined to form fused heterocycle-alkylring systems. Substituents including heterocyclic groups (e.g.,heteroaryloxy, and heteroaralkylthio) are defined by analogy to theabove-described terms.

The term “heterocycloalkyl” as used herein refers to a heterocycle groupthat is joined to a parent structure by one or more alkyl groups asdescribed above, e.g., 2-piperidylmethyl, and the like. The term“heterocycloalkyl” refers to a heteroaryl group that is joined to aparent structure by one or more alkyl groups as described above, e.g.,2-thienylmethyl, and the like.

For brevity, the term “aryl” when used in combination with other terms(e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroarylrings as defined above. Thus, the term “arylalkyl” is meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) including thosealkyl groups in which a carbon atom (e.g., a methylene group) has beenreplaced by, for example, an oxygen atom (e.g., phenoxymethyl,2-pyridyloxymethyl, 3-(1-naphthyloxy)propyl, and the like).

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “aryl” and“heteroaryl”) includes both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) are generically referred to as “alkyl groupsubstituents,” and they can be one or more of a variety of groupsselected from, but not limited to: —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR',-halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂ in a number ranging from zero to (2 m′+1), where m′ is the totalnumber of carbon atoms in such radical. R′, R″, R′″ and R″″ eachpreferably independently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, e.g., aryl substitutedwith 1-3 halogens, substituted or unsubstituted alkyl, alkoxy orthioalkoxy groups, or arylalkyl groups. When a compound of the inventionincludes more than one R group, for example, each of the R groups isindependently selected as are each R′, R″, R′″ and R″″ groups when morethan one of these groups is present. When R′ and R″ are attached to thesame nitrogen atom, they can be combined with the nitrogen atom to forma 5-, 6-, or 7-membered ring. For example, —NR′R″ is meant to include,but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the abovediscussion of substituents, one of skill in the art will understand thatthe term “alkyl” is meant to include groups including carbon atoms boundto groups other than hydrogen groups, such as haloalkyl (e.g., —CF₃ and—CH₂CF₃) and acyl (e.g., —C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and thelike).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are generically referredto as “aryl group substituents.” The substituents are selected from, forexample: halogen, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, -halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —CN and—NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy, and fluoro(C₁-C₄)alkyl,in a number ranging from zero to the total number of open valences onthe aromatic ring system; and where R′, R″, R′″ and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted aryl and substituted or unsubstituted heteroaryl. When acompound of the invention includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″and R″″ groups when more than one of these groups is present. In theschemes that follow, the symbol X represents “R” as described above.

The aryl and heteroaryl substituents described herein are unsubstitutedor optionally and independently substituted by H, halogen, cyano,sulfonic acid, carboxylic acid, nitro, alkyl, perfluoroalkyl, alkoxy,alkylthio, amino, monoalkylamino, dialkylamino or alkylamido.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally be replaced with a substituent of the formula-T-C(O)—(CRR′)_(d)-U-, wherein T and U are independently —NR—, —O—,—CRR′— or a single bond, and q is an integer of from 0 to 3.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula -A-(CH₂)_(r)-B-, wherein A and B are independently —CRR′—, —O—,—NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′— or a single bond, and r is aninteger of from 1 to 4. One of the single bonds of the new ring soformed may optionally be replaced with a double bond. Alternatively, twoof the substituents on adjacent atoms of the aryl or heteroaryl ring mayoptionally be replaced with a substituent of the formula—(CRR′)_(s)—X—(CR″R′″)_(d)—, where s and d are independently integers offrom 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or —S(O)₂NR′—.The substituents R, R′, R″ and R′″ are preferably independently selectedfrom hydrogen or substituted or unsubstituted (C₁-C₆)alkyl.

As used herein, the term “heteroatom” includes oxygen (O), nitrogen (N),sulfur (S), phosphorus (P) and silicon (Si).

The term “amino” or “amine group” refers to the group —NR′R″ (or NRR′R″)where R, R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted aryl alkyl, heteroaryl, and substituted heteroaryl. Asubstituted amine being an amine group wherein R′ or R″ is other thanhydrogen. In a primary amino group, both R′ and R″ are hydrogen, whereasin a secondary amino group, either, but not both, R′ or R″ is hydrogen.In addition, the terms “amine” and “amino” can include protonated andquaternized versions of nitrogen, comprising the group —NRR′R″ and itsbiologically compatible anionic counterions.

The term “attachment site” as used herein refers to a site on a moietyor a molecule, e.g. a quencher, a fluorescent dye, an avidin, or anantibody, to which is covalently attached, or capable of beingcovalently attached, to a linker or another moiety. The term “aqueoussolution” as used herein refers to a solution that is predominantlywater and retains the solution characteristics of water. Where theaqueous solution contains solvents in addition to water, water istypically the predominant solvent.

The term “BAPTA” as used herein refers to a metal-chelating compoundthat is 1,2-bis(2-aminophenoxy)ethane-N,N,N′N′-tetraacetic acid or itsanalogs, derivatives, ring-fused variants and conjugates, and allmetallic and nonmetallic salts, partial salts and hydrates thereof,including any corresponding compounds disclosed in U.S. Pat. Nos.4,603,209; 4,849,362; 5,049,673; 5,453,517; 5,459,276; 5,516,911;5,501,980; 6,162,931 and 5,773,227 (supra). When used generically,“BAPTA” refers to two benzene rings that are joined by a C₁-C₃hydrocarbon bridge terminated by oxygen atoms, including methylenedioxy(—OCH₂O—), ethylenedioxy (—OCH₂CH₂O—) or propylenedioxy (—OCH₂CH₂CH₂O—)bridging groups, where each benzene ring is optionally substituted byone or more substituents that adjust the metal ion-binding affinity,solubility, chemical reactivity, spectral properties or other physicalproperties of the compound. In a preferred embodiment of the presentinvention “BAPTA” is covalently attached to a chemical moiety A that, incombination with an appropriate trivalent metal ion and an acid, permitsdetection or isolation of phosphorylated target molecules as a ternarycomplex. BAPTA derivatives additionally include compounds in which thebenzene rings of the BAPTA structure are substituted by or fused toadditional aromatic, or heteroaromatic rings.

The term “Linker” or “L”, as used herein, refers to a single covalentbond or a series of stable covalent bonds incorporating 1-30 nonhydrogenatoms selected from the group consisting of C, N, O, S and P thatcovalently attach the phosphate-binding compounds to another moiety suchas a chemically reactive group or a phosphorylated target molecule.Exemplary linking members include a moiety that includes —C(O)NH—,—C(O)O—, —NH—, —S—, —O—, and the like. A “cleavable linker” is a linkerthat has one or more cleavable groups that may be broken by the resultof a reaction or condition. The term “cleavable group” refers to amoiety that allows for release of a portion, e.g., a label orphosphorylated target molecule, of a conjugate from the remainder of theconjugate by cleaving a bond linking the released moiety to theremainder of the conjugate. Such cleavage is either chemical in nature,or enzymatically mediated. Exemplary enzymatically cleavable groupsinclude natural amino acids or peptide sequences that end with a naturalamino acid.

In addition to enzymatically cleavable groups, it is within the scope ofthe present invention to include one or more sites that are cleaved bythe action of an agent other than an enzyme. Exemplary non-enzymaticcleavage agents include, but are not limited to, acids, bases, light(e.g., nitrobenzyl derivatives, phenacyl groups, ortho-hydroxcinnamateesters, benzoin esters), and heat. Many cleaveable groups are known inthe art. See, for example, Jung et al., Biochem. Biophys. Acta, 761:152-162 (1983); Joshi et al., J. Biol. Chem., 265: 14518-14525 (1990);Zarling et al., J. Immunol., 124: 913-920 (1980); Bouizar et al., Eur.J. Biochem., 155: 141-147 (1986); Park et al., J. Biol. Chem., 261:205-210 (1986); Browning et al., J. Immunol., 143: 1859-1867 (1989).Moreover a broad range of cleavable, bifunctional (both homo- andhetero-bifunctional) spacer arms are commercially available.

An exemplary cleavable group, an ester, is cleavable group that may becleaved by a reagent, e.g. sodium hydroxide, resulting in acarboxylate-containing fragment and a hydroxyl-containing product.

The linker can be used to attach the compound to another component of aconjugate, such as a targeting moiety (e.g., antibody, ligand,non-covalent protein-binding group, etc.), an analyte, a biomolecule, adrug and the like.

The term “metal chelator” or “metal-chelating moiety” as used hereinrefers to a chemical moiety that combines with a metal ion to form achelate ring structure. For the purposes of the present invention themetal chelator has affinity for a metal ion that has simultaneousaffinity for the metal chelator and a phosphate target molecule in amoderately acidic environment. Examples of metal-chelating moietiesinclude, but are not limited to, BAPTA, IDA, DTPA and phenanthroline.The metal chelators are optionally substituted by substituents thatadjust the ion-binding affinity, solubility, chemical reactivity,spectral properties or other physical properties of the compoundprovided that the metal chelator is not sulfonated.

The term “photoactivatable reactive group” as used herein refers to achemical moiety that becomes chemically active by exposure to anappropriate wavelength, typically a UV wavelength. Once activated thereactive group is capable of forming a covalent bond with a proximalmoiety on a biological or non-biological component.

The terms “protein” and “polypeptide” are used herein in a generic senseto include polymers of amino acid residues of any length. The term“peptide” is used herein to refer to polypeptides having less than 100amino acid residues, typically less than 15 amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The peptide or protein may be further conjugated to orcomplexed with other moieties such as dyes, haptens, radioactiveisotopes, natural and synthetic polymers (including microspheres),glass, metals and metallic particles, proteins and nucleic acids.

The term “reactive group” as used herein refers to a group that iscapable of reacting with another chemical group to form a covalent bond,i.e. is covalently reactive under suitable reaction conditions, andgenerally represents a point of attachment for another substance. Thereactive group is a moiety, such as a photoactivatable group, carboxylicacid or succinimidyl ester, on the compounds of the present inventionthat is capable of chemically reacting with a functional group on adifferent compound to form a covalent linkage resulting in aphosphate-binding labeled component. Reactive groups generally includenucleophiles, electrophiles and photoactivatable groups.

Exemplary reactive groups include, but not limited to, olefins,acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes,ketones, carboxylic acids, esters, amides, cyanates, isocyanates,thiocyanates, isothiocyanates, amines, hydrazines, hydrazones,hydrazides, diazo, diazonium, nitro, nitriles, mercaptans, sulfides,disulfides, sulfoxides, sulfones, sulfonic acids, sulfinic acids,acetals, ketals, anhydrides, sulfates, sulfenic acids isonitriles,amidines, imides, imidates, nitrones, hydroxylamines, oximes, hydroxamicacids thiohydroxamic acids, allenes, ortho esters, sulfites, enamines,ynamines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates,imines, azides, azo compounds, azoxy compounds, and nitroso compounds.Reactive functional groups also include those used to preparebioconjugates, e.g., N-hydroxysuccinimide esters, maleimides and thelike. Methods to prepare each of these functional groups are well knownin the art and their application to or modification for a particularpurpose is within the ability of one of skill in the art (see, forexample, Sandler and Karo, eds. ORGANIC FUNCTIONAL GROUP PREPARATIONS,Academic Press, San Diego, 1989).

As used herein the term “sulfonic acid” means either —SO₃H, or a salt ofsulfonic acid. Also as used herein the term “carboxylic acid” meanseither —COOH, or a salt of carboxylic acid. Appropriate salts ofsulfonic and carboxylic acids include, among others, K⁺, Na⁺, Cs⁺, Li⁺,Ca²⁺, Mg²⁺, ammonium, alkylammonium or hydroxyalkylammonium salts, orpyridinium salts. Alternatively, the counterion of the sulfonic acid orcarboxylic acid may form an inner salt with a positively charged atom onthe dye itself, typically a quaternary nitrogen atom.

The Compounds

The synthetic precursors have the formula:

G represents the atoms necessary to form a 5- or 6-membered aromatic orheteroaromatic fused ring, that is optionally substituted one or moretimes by sulfonic acid, carboxylic acid, or C₁-C₆ alkyl or alkoxy thatis optionally substituted by carboxylic acid, sulfonic acid, or halogen;or by an aryl or heteroaryl ring that is optionally substituted one ormore times by C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, cyano, halogen, azido,carboxylic acid, sulfonic acid, or halomethyl. The fused ring Goptionally contains one or two additional fused aromatic orheteroaromatic rings that are optionally sulfonated one or more times.

Suitable examples of fused aromatic rings include, but are not limitedto, substituted or unsubstituted derivatives of benzenes, naphthalenes,anthracenes, phenanthracenes, or pyrenes. Preferred aromatic ringsinclude substituted or unsubstituted benzene or naphthalenes.

Suitable examples of fused heteroaromatic rings include, but are notlimited to, furans, thiophenes, pyrrols, benzofurans, benzothiophenes,indoles, pyridines, quinolines, isoquinolines, oxazoles, benzoxazoles,thiazoles, benzothiazoles, isoxazoles, imidazoles, benzimidazoles,naphthofurans, naphthothiophenes, benzindoles, benzoquinolines, oracridines. Preferred fused heteroaromatic rings include substituted orunsubstituted one or more of R¹, R², and R⁶ is an aryl or heteroarylring that is optionally substituted one or more times by C₁-C₆ alkyl,C₁-C₆ perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonicacid, or halomethylpyridines, thiophenes, pyrroles, indoles, oxazoles,benzothiophenes, and benzoxazoles.

R⁶ is H, cyano, halogen, carboxylic acid, or sulfonic acid, or a C₁-C₆alkyl or C₁-C₆ alkoxy that is optionally substituted by carboxylic acid,sulfonic acid, or halogen. Additionally R⁶ is an aryl or heteroaryl ringthat is optionally substituted one or more times by C₁-C₆ alkyl, C₁-C₆perfluoroalkyl, cyano, halogen, azido, carboxylic acid, sulfonic acid,or halomethyl.

The substituents R³ and R⁴ are independently H, or a C₁-C₆ alkyl that isoptionally substituted by carboxylic acid, sulfonic acid, amino,hydroxy, or halogen. Alternatively one or both of R³ and R⁴ isindependently an aromatic or heteroaromatic ring that is optionallysubstituted one or more times by C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, orhalomethyl. In another aspect of the invention, R³ and R⁴, taken incombination, form a 5- or 6-membered ring that optionally contains 1 or2 heteroatoms. Typically, where R³ and R⁴ form a ring, it is analicyclic ring. R³ and R⁴ are each typically alkyl, and preferably R³and R⁴ are methyl.

The R⁵ substituent is H, methyl, carboxymethyl, or a C₂-C₆ alkyl that isoptionally substituted by carboxylic acid, sulfonic acid, amino, orhalogen. Alternatively, R⁵ is an aryl or heteroaryl ring that isoptionally substituted one or more times by C₁-C₆ alkyl, C₁-C₆perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, orhalomethyl. In another aspect of the invention, R⁴ taken in combinationwith R⁵, or R⁵ taken in combination with R⁶, forms a 5- or 6-memberedalicyclic ring.

The R⁷ substituent is hydrogen, C₁-C₆ alkyl, or C₁-C₆ alkoxy. Typically,R⁷ is hydrogen.

Y is optionally H, OH, NH₂, NO, —(CO)—R⁹, or —(CO)—O—R¹⁰, where R⁹ andR¹⁰ are H, C₁-C₆ alkyl, a substituted or unsubstituted aryl orheteroaryl ring system having 1-2 rings.

Z is optionally H, OH, NHR¹⁷, SH, or C(CR¹¹R¹²)₂OH; where R¹⁷ is a C₁-C₆alkyl that is optionally substituted by carboxylic acid, sulfonic acid,amino, or halogen. The R¹¹ and R¹² substituents are independently C₁-C₆alkyls that are themselves optionally substituted by carboxylic acid,sulfonic acid, or halogen, or R¹¹ and R¹² taken in combination form a 5-or 6-membered alicyclic ring. Preferably R¹¹ and R¹² are each methyl.

In one embodiment, the synthetic precursor has the formula

where R¹ and R² are independently selected from H, cyano, halogen,carboxylic acid, or sulfonic acid, or one or more of R¹ and R² may be aC₁-C₆ alkyl or alkoxy that is optionally substituted by carboxylic acid,sulfonic acid, or halogen. Additionally one or more of R¹ and R² is anaryl or heteroaryl ring that is optionally substituted one or more timesby C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, cyano, halogen, azido, carboxylicacid, sulfonic acid, or halomethyl. In another aspect of the invention,R¹ in combination with R² forms a fused aromatic or heteroaromatic ringthat is optionally sulfonated one or more times. In another aspect ofthe invention, R² taken in combination with R³ forms a 5- or 6-memberedalicyclic ring.

One of X and E is selected from O, S, NR⁸, or CR^(1′)═CR^(2′), and theother is absent. The substituent R⁸ is H, methyl, carboxymethyl, or aC₂-C₆ alkyl that is optionally substituted by carboxylic acid, sulfonicacid, amino, or halogen. The substituents R^(1′) and R^(2′) are asdefined above for R¹ and R². In one embodiment, one of X and E is S orO. In another embodiment, X is O, S, NR⁸, or CR^(1′)═CR^(2′), and E isabsent. In yet another embodiment, E is O or S, and X is absent.

Y is optionally H, OH, NH₂, NO, —(CO)—R⁹, or —(CO)—O—R¹⁰, where R⁹ andR¹⁰ are H, C₁-C₆ alkyl, a substituted or unsubstituted aryl orheteroaryl ring system having 1-2 rings.

Z is optionally H, OH, NHR¹⁷, SH, or C(CR¹¹R¹²)₂OH; where R¹⁷ is a C₁-C₆alkyl that is optionally substituted by carboxylic acid, sulfonic acid,amino, or halogen. The R¹¹ and R¹² substituents are independently C₁-C₆alkyls that are themselves optionally substituted by carboxylic acid,sulfonic acid, or halogen, or R¹¹ and R¹² taken in combination form a 5-or 6-membered alicyclic ring.

The precursors of the invention are optionally substituted by acovalently bound reactive group (-L-R_(x)) or conjugated substance(-L-S_(c)) as will be described below. In this embodiment, one or moreof R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ is -L-R_(x) or -L-S_(c), or where R¹taken in combination with R² forms a fused aromatic or heteroaromaticring, the resulting ring is substituted by -L-R_(x) or -L-S_(c).

Condensation Reactions of the Novel Precursors

A variety of useful and novel dyes are readily prepared using theprecursors of the invention. The dyes of the invention are prepared bycondensing a first precursor (a precursor of the invention) with asecond precursor, simultaneous with, before, or after reaction with athird precursor. The types of precursors and possible dye products aredescribed in greater detail below. The utilization of the precursors ofthe invention in these syntheses results in dyes whose absorption isshifted to substantially longer wavelengths, compared to structurallyrelated dyes that do not possess the additional fused ring systems.

Derivatives of 3-aminophenol are valuable synthetic intermediates in thesynthesis of a variety of dyes. Numerous classes of commerciallyimportant dyes may be prepared with appropriately substituted3-minophenols using well known synthetic strategies. In particular,3-aminophenols have been utilized in the preparation of coumarin dyes(U.S. Pat. No. 5,696,157), rhodol dyes (U.S. Pat. No. 5,227,487),rhodamine dyes (U.S. Pat. No. 6,130,101), and oxazine dyes.3-Aminophenols have also been utilized to prepare annelated rhodol dyes(seminaphthorhodafluor dyes) commercially available under the trademarkSNARF (U.S. Pat. No. 4,945,171).

Typically the condensation reaction utilizing the 3-aminophenolderivatives of the invention is acid-catalyzed, and utilizes a firstprecursor, a second precursor, and optionally utilizes a thirdprecursor. This condensation occurs in the presence or absence ofvarious acid catalysts (such as zinc chloride, p-toluenesulfonic acid,sulfuric acid, or methanesulfonic acid). An aqueous workup, typicallyfollowed by column chromatography, yields the desired dye.

The first precursor is a derivative of 3-aminophenol of the inventionhaving the formula

wherein G, R³, R⁴, R⁵, and R⁶ are as defined earlier. R⁷ is hydrogen orC₁-C₆ alkyl. Y is optionally H, OH, NO, —(CO)—R⁹, or —(CO)—O—R¹⁰, whereR⁹ and R¹⁰ are H, C₁-C₆ alkyl, a substituted or unsubstituted aryl orheteroaryl ring system having 1-2 rings.

Typically, the first precursor has the formula

wherein E, X, R¹, R², R³, R⁴, R⁵, and R⁶ are as defined earlier. R⁷ ishydrogen or C₁-C₆ alkyl. Y is optionally H, OH, NO, —(CO)—R⁹, or—(CO)—O—R¹⁰, where R⁹ and R¹⁰ are H, C₁-C₆ alkyl, a substituted orunsubstituted aryl or heteroaryl ring system having 1-2 rings.

Where Y is NO, the first precursor is a 6-nitroso-3-aminophenolderivative. Where Y is —(CO)—R⁹, the first precursor is a6-acyl-3-aminophenol derivative.

The second precursor is permitted to be a 3-aminophenol (to yield arhodamine), a resorcinol (to yield a rhodol), a 1,6-naphthalenediol (toyield an annelated rhodol), a 6-amino-1-naphthol (to yield an annelatedrhodamine), an alpha-methylene acid, an alpha methylene ester, analpha-methylene nitrile, or a beta-keto ester (all of which yieldcoumarins). Generic examples of each of these types of precursors aregiven below, although a variety of substitutions and variations arepermissible without effectively diminishing the efficacy of thecondensation reaction:

The condensation reaction optionally incorporates a third precursor. Thethird precursor is typically an aldehyde or an acid, including diacids,acid anhydrides, esters, acid halides and similar acylating agentsderived from acids. Examples of aldehydes include formaldehyde, loweraliphatic and alicyclic aldehydes, heterocyclic aldehydes and especiallybenzaldehydes and less preferably naphthaldehydes. Most commonly thethird precursor is an aliphatic or aromatic diacid, or a cyclicanhydride of an aliphatic or aromatic diacid. Particularly preferredthird precursors are derivatives of succinic acid, glutaric acid,phthalic acid, or ortho-sulfobenzoic acid.

In those condensation reactions where the third precursor is present,the reaction of the first, second and third precursors is typically astepwise reaction. The third precursor reacts with either the first orsecond precursor to form an intermediate condensation product, whichthen reacts with the remaining precursor.

Where the first and second precursors are the same, the resulting dye issymmetric. Typically, where symmetric dyes are prepared, thecondensation includes a third precursor, as described above. Commonlyone molecule of a substituted or unsubstituted phthalic acid oranhydride or a substituted or unsubstituted ortho-sulfobenzaldehyde orortho-sulfobenzoic acid (the third precursor) is condensed with at leasttwo molecules of a 3-aminophenol of the invention (first and secondprecursor, which are the same) to produce a symmetric rhodamine dye.Less commonly a 3-aminophenol of the invention (first precursor) and aresorcinol or a different 3-aminophenol (second precursor) is condensedwith a substituted or unsubstituted phthalic acid or o-sulfobenzoic acidderivative (third precursor) to produce rhodol or asymmetric rhodaminedyes of the invention.

Where the first precursor is a 6-nitroso-3-aminophenol, and the secondprecursor is a resorcinol or a 3-aminophenol, the resulting dye is anoxazine. Alternatively, the second precursor is nitroso-substituted,such as a 4-nitrosoresorcinol or a 6-nitroso-3-aminophenol. In eithercase, the resulting product is an oxazine dye.

To prepare coumarin dyes, a first precursor that is a6-acyl-3-aminophenol is reacted with a second precursor that is analpha-methylene acid, ester, or nitrile to yield a 3-substitutedcoumarin, the nature of the 3-substituent of the resulting coumarin dyebeing dependent upon the 6-acyl moiety on the first precursor.4-Substituted coumarin dyes are prepared using a first precursor that isa 3-aminophenol of the invention with a second precursor that is abeta-keto ester. In this instance, the nature of the 4-substituent isdependent upon beta-keto ester selected.

Selected condensation reactions of the invention are outlined in Table1, below.

TABLE 1 First Precursor Second Precursor Third Precursor Product DyeExamples a 3-aminophenol a 3-aminophenol an aldehyde, acid, diacid, asymmetric or asymmetric 18, 29, 30, 32, or anhydride rhodamine 33, 36 a3-aminophenol a 6-acyl-3-aminophenol none a 6-acyl-3- a 3-aminophenolnone aminophenol a 3-aminophenol a resorcinol an aldehyde, acid, diacid,a rhodol 35 or anhydride a 6-acyl-3- a resorcinol none aminophenol a3-aminophenol a 4-acylresorcinol none a 3-aminophenol a4-nitrosoresorcinol, or none an oxazine 21 a 6-nitroso-3- aminophenol a6-nitroso-3- a resorcinol, or none aminophenol a 3-aminophenol a3-aminophenol a 1,6-naphthalenediol, or an aldehyde, acid, diacid, anannelated rhodol or 38 6-amino-1-naphthol or anhydride rhodamine a6-nitroso-3- a 1,6-naphthalenediol, or none an annelated oxazineaminophenol a 6-amino-1-naphthol a 6-acyl-3- an alpha-methylene acid,none a 3-substituted coumarin 20 aminophenol ester or nitrile a3-aminophenol a beta-keto ester none a 4-substituted coumarin

In addition to the rhodamine, annelated rhodamine, rhodol, annelatedrhodol, oxazine, annelated oxazine and coumarin dyes whose synthesis andproperties is described in this invention, first precursors wherein Z isnot hydroxy are useful synthetic precursors a variety of analogousclasses of dyes. Precursors wherein Z is SH are useful for preparingthiocoumarins, thiorhodamines, thiooxazines and thiorhodols (for exampleas in EP 0 330 444 to Chen et al., (1989)) and their annelated versions.Precursors wherein Z is NHR¹⁷ are useful for preparing “azacoumarins”(carbostyryls), “azarhodamines” (acridines), “azarhodols” (acridines),“azaoxazines” (phenazines) and their annelated versions. Precursorswherein Z is C(CR¹¹R¹²)₂OH are useful for preparing carbazine dyes andtheir analogs. Precursors wherein Z is H are useful for preparingtriarylmethane dyes and their analogs (Example 34).

In one aspect of the invention, the resulting dye is sulfonated.Sulfonation can be done subsequent to the condensation reaction (as inExample 23), or one or more precursors may be sulfonated prior toformation of the dye (as for Compound 34 in Example 18).

Sulfonation of dyes is typically carried out by stirring the dye infuming sulfuric acid (20-30% SO₃ content) or concentrated sulfuric acidat an appropriate temperature. Mono-sulfonation of rhodol dyes iscarried out by stirring the appropriate rhodol dye in fuming sulfuricacid at 0° C. for several hours. Bis-sulfonation of rhodols at both the4′- and 5′- positions, if available, is achieved by stirring the dye infuming sulfuric acid at room temperature for several hours. Sulfonationof most rhodamine or rosamine dyes at the 4′- and 5′- positions, ifavailable, is carried out with fuming sulfuric acid at 0° C.; thesulfonation is usually complete as soon as a homogeneous solution isachieved during stirring.

Post-condensation modification of xanthene-based dyes is well known. Forexample, the xanthene portion of the dye can be halogenated by treatmentwith the appropriate halogenating agent, such as liquid bromine.Xanthenes containing unsaturated fused rings can be hydrogenated to thesaturated derivatives. When trimellitic anhydride, nitrophthalicanhydride, or their derivatives are used in the dye synthesis, twoisomeric carboxylates or nitro derivatives are typically formed. Theseisomers are separated or, in most cases, used as the mixture of isomers.The reduced derivatives of xanthylium dyes are typically prepared bychemical reduction of the xanthenone portion with zinc dust, borohydridein organic solvents, or by catalytic hydrogenation. The amino andhydroxyl groups of the dyes of the invention can be acylated oralkylated to yield amides, esters and ethers. Selected amide, ether andester derivatives of the invention possess potential utility aschromogenic or fluorogenic enzyme substrates.

The selection of an appropriate polyhalogenated phthalic acid derivativeor benzaldehyde in the condensation of the xanthylium dye results in adye having a tetra- or pentachlorinated or tetra- or pentafluorinatedphenyl ring at the 9-position. These polyhaloaryl substituted dyes havebeen shown to react with thiols via a displacement reaction, and therebyprovide a facile method of introducing additional reactive groups(Example 19; and as discussed by Gee, et al. TET. LETT. 37, 7905(1996)).

The dihydroxanthene and xanthylium versions of the dyes of the inventionare freely interconvertible by well-known oxidation or reductionreagents, including borohydrides, aluminum hydrides, hydrogen/catalyst,and dithionites. A variety of oxidizing agents mediate the oxidation ofdihydroxanthenes, including molecular oxygen in the presence or absenceof a catalyst, nitric oxide, peroxynitrite, dichromate,triphenylcarbenium and chloranil. The xanthenes are also oxidized byenzyme action, including horseradish peroxidase in combination withperoxides. Dihydroxanthene dye precursors may also be oxidized insidecertain living cells, yielding the corresponding dye compound.

Examples of synthetic strategies for selected dyes of the invention, aswell as their characterization, synthetic precursors, conjugates andmethod of use are provided in the examples below.

Selected Dye Embodiments

In one aspect of the invention, the dyes of the invention that resultfrom the condensation reaction with the precursors described above havethe formula

wherein G, R³, R⁴, R⁶, and R⁷ are as described above for the novelprecursors of the invention.

The Q moiety is N or CR²⁸, wherein R²⁸ is H, F, CN, a carboxylic acid, asalt of carboxylic acid, or a carboxylic acid ester of a C₁-C₆ alcohol.Alternatively R²⁸ is a C₁-C₆ alkyl that is optionally substituted one ormore times by carboxylic acid, sulfonic acid, amino, or halogen. Inanother preferred embodiment, Q is CR²⁸ where R²⁸ has the formula

where the substituents R³⁰, R³¹, R³², R³³ and R³⁴ are independently H,F, Cl, Br, I, sulfonic acid, carboxylic acid, CN, nitro, hydroxy, azido,amino, hydrazino, C₁-C₁₈ alkyl, C₁-C₁₈ alkoxy, C₁-C₁₈ alkylthio, C₁-C₁₈alkanoylamino, C₁-C₁₈ alkylaminocarbonyl, C₂-C₃₆ dialkylaminocarbonyl,C₁-C₁₈ alkyloxycarbonyl, or C₇-C₁₈ arylcarboxamido. Where any of theR³⁰, R³¹, R³², R³³ and R³⁴ substituents are alkyl, alkoxy, alkylthio,alkanoylamino, alkylaminocarbonyl, dialkylaminocarbonyl,alkyloxycarbonyl, or arylcarboxamido, the alkyl or aryl portions of thesubstituents are optionally substituted one or more times by F, Cl, Br,I, hydroxy, carboxylic acid, a carboxylic acid ester of a C₁-C₆ alcohol,sulfonic acid, amino, alkylamino, dialkylamino or alkoxy (the alkylportions of each having 1-6 carbons). Alternatively, one pair ofadjacent substituents R³¹ and R³², R³² and R³³ or R³³ and R³⁴, whentaken in combination, form a fused 6-membered aromatic ring that isoptionally further substituted by carboxylic acid. Alternatively, one ormore of R³⁰, R³¹, R³², R³³ and R³⁴ is -L-R_(x) or -L-S_(c), as describedbelow. In a preferred embodiment, at least one of R³⁰, R³¹, R³², R³³ andR³⁴ is a -L-S_(c), wherein L is a single covalent bond and Sc is a metalchelating moiety.

In one aspect of the invention, the resulting dye is a coumarin. In thisembodiment, A is O, and D is a monovalent substituent. In another aspectof the invention, A and D, when taken in combination, form an aromaticor heteroaromatic ring system having 1-3 additional rings, where thering system is optionally substituted.

In one aspect of the invention, the dye of the invention is a coumarindye having the formula

where GR³, R⁴, R⁵, and R⁶ are as defined above.

The substituents R¹⁵ and R¹⁶ are independently hydrogen, cyano, nitro,halogen, carboxylic acid, sulfonic acid, or a C₁-C₆ alkyl that isoptionally substituted by carboxylic acid, sulfonic acid, or halogen.Alternatively, one or more of R¹⁵ and R¹⁶ is an aromatic orheteroaromatic ring system having 1-2 fused rings that is optionallysubstituted one or more times by C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl,cyano, halogen, carboxylic acid, sulfonic acid, or halomethyl. Inanother aspect of the invention, one of R¹⁵ and R¹⁶ is -L-R_(x) or-L-S_(c).

In one aspect of the invention, one of R¹⁵ and R¹⁶ is nonhydrogen. Inanother aspect of the invention, both R¹⁵ and R¹⁶ are nonhydrogen. Inone aspect of the invention, R¹⁶ is H. In another aspect of theinvention, R¹⁶ is chloromethyl or bromomethyl. In yet another aspect ofthe invention, R⁵ is not hydrogen, and R⁶ is methyl or C₁-C₆ alkyloptionally substituted by sulfonic acid or carboxylic acid. Where one ofR¹⁵ or R¹⁶ is a heteroaromatic ring system, it is typically abenzothiazole.

In another aspect of the invention, the dye of the invention is aderivative of a xanthene or oxazine dye having the formula

where G, R³, R⁴, R⁵, and R⁶ are as defined above.

The substituents R²⁰ and R²¹ are hydrogen, cyano, halogen, carboxylicacid, sulfonic acid, or a C₁-C₆ alkyl or C₁-C₆ alkoxy that is itselfoptionally substituted by carboxylic acid, sulfonic acid, or halogen.Alternatively, one or both of R²⁰ and R²¹ is an aromatic orheteroaromatic ring that is optionally substituted one or more times byC₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, cyano, halogen, carboxylic acid,sulfonic acid, or halomethyl. Either or both of R²⁰ and R²¹ isoptionally -L-R_(x); or -L-S_(c).

The J moiety is O or NR³⁷R³⁸, where R³⁷ and R³⁸ are independently H or aC₁-C₆ alkyl that is optionally substituted by carboxylic acid, sulfonicacid, amino, or halogen. Alternatively, one or more of R³⁷ and R³⁸ is-L-R_(x) or -L-S_(c).

Alternatively one of R³⁷ and R³⁸ is an aryl or heteroaryl ring, or R³⁷when taken in combination with R³⁸ forms a saturated 5- or 6-memberedheterocycle that is a piperidine, a morpholine, a pyrrolidine or apiperazine, wherein the heterocycle is optionally substituted by methyl,carboxylic acid, or a carboxylic acid ester of a C₁-C₆ alkyl.

In yet another alternative, R³⁷ taken in combination with R²⁰, or R³⁸taken in combination with R²¹, or both, form a 5- or 6-membered ringthat is saturated or unsaturated, and is optionally substituted by oneor more sulfonic acids, or by one or more C₁-C₆ alkyl groups that areoptionally substituted by sulfonic acid.

Q is N or CR²⁸, as described above.

In another aspect of the invention, the dye of the invention is aseminaphthorhodafluor derivative having the formula

where G, R³, R⁴, R⁵, R⁶, and Q are as defined above.

The substituents R²¹, R²³, R²⁴, and R²⁵ are hydrogen, cyano, nitro,halogen, carboxylic acid, or sulfonic acid, or a C₁-C₆ alkyl that isoptionally substituted by carboxylic acid, sulfonic acid, or halogen.Alternatively one or more of R²¹, R²³, R²⁴, and R²⁵ is an aromatic orheteroaromatic ring that is optionally substituted one or more times byC₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, cyano, halogen, carboxylic acid,sulfonic acid, or halomethyl. Additionally, one or more of R²¹, R²³,R²⁴, and R²⁵ is -L-R_(x) or -L-S_(c).

The J moiety is O or NR³⁷R³⁸, where R³⁷ and R³⁸ are independently H or aC₁-C₆ alkyl that is optionally substituted by carboxylic acid, sulfonicacid, amino, or halogen. Alternatively, one or more of R³⁷ and R³⁸ is-L-R_(x) or -L-S_(c).

Derivatives of seminaphthorhodafluor are typically useful as fluorescentpH indicators (see Example 44).

In yet another aspect of the invention, the dye product incorporates twoprecursor compounds of the invention to yield a rhodamine dye having theformula

where G, R³, R⁴, R⁵, R⁶, and Q are as defined above.

The substituent R⁴⁶ is H, cyano, halogen, carboxylic acid, sulfonicacid, or C₁-C₆ alkyl or alkoxy that is optionally substituted bycarboxylic acid, sulfonic acid, or halogen. Alternatively R⁴⁶ is an arylor heteroaryl ring that is optionally substituted one or more times byC₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, cyano, halogen, azido, carboxylicacid, sulfonic acid, or halomethyl. R⁴⁶ may also be -L-R_(x) or-L-S_(c).

The substituents R⁴³ and R⁴⁴ are independently H, or a C₁-C₆ alkyl thatis optionally substituted by carboxylic acid, sulfonic acid, amino,hydroxy, or halogen. Alternatively one or both of R⁴³ and R⁴⁴ isindependently an aromatic or heteroaromatic ring that is optionallysubstituted one or more times by C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, orhalomethyl. R⁴³ and R⁴⁴ are each typically alkyl, and preferably R⁴³ andR⁴⁴ are methyl. In another aspect of the invention, R⁴³ taken incombination with R⁴⁴, forms a 5- or 6-membered alicyclic ring.Alternatively, R⁴³ and R⁴⁴ are -L-R_(x) or -L-S_(c).

The R⁴⁵ substituent is H, methyl, carboxymethyl, or a C₂-C₆ alkyl thatis optionally substituted by carboxylic acid, sulfonic acid, amino, orhalogen. Alternatively, R⁴⁵ is an aryl or heteroaryl ring that isoptionally substituted one or more times by C₁-C₆ alkyl, C₁-C₆perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, orhalomethyl. In another aspect of the invention, R⁴⁴ taken in combinationwith R⁴⁵, or R⁴⁵ taken in combination with R⁴⁶, forms a 5- or 6-memberedalicyclic ring. Alternatively, R⁴⁵ is -L-R_(x) or -L-S_(c).

The fused ring W is a 5- or 6-membered aromatic or heteroaromatic fusedring that has the same parameters defined for G above, and is optionallythe same as or different from G.

In one embodiment, the fused ring W has the formula

where X′ and E′ are defined as for X and E above, and R⁴¹ is defined asR¹ above, and R⁴² is defined as R² above.

Typically, in this embodiment, the dye is fully symmetrical. That is, Gand W are the same. In particular, X=X′, E=E′, R¹=R⁴¹, and R²=R⁴². In afully symmetrical dye, R³ and R⁴³ are the same, R⁴ and R⁴⁴ are the same,R⁵ and R⁴⁵ are the same, and R⁶ and R⁴⁶ are the same. In another aspectof the invention, R³, R⁴, R⁴³, and R⁴⁴ are each methyl. In one preferredembodiment, each X is S, and each R¹ is sulfonic acid.

In one embodiment, the dyes of the invention are derivatives of 3-and/or 6-amino xanthenes that are substituted at one or more aminonitrogen atoms by an aromatic or heteroaromatic ring system (i.e., oneor more of R⁵, R³⁷, and R³⁸ is an aromatic or heteroaromatic ring).N-aryl rhodamines have been shown to be efficient and minimallyfluorescent energy acceptors (U.S. Pat. No. 6,399,392), and suchcompounds are useful in any application where a quenching energyacceptor is useful, particularly in applications utilizing fluorescenceresonance energy transfer (FRET). In a preferred embodiment, one or moreof R⁵, R³⁷ and R³⁸ is phenyl.

In another embodiment of the invention, the dyes have the formula:

wherein R³, R⁴, R⁵, R⁴³, R⁴⁴, and R⁴⁵ are independently methyl or ethyl;

-   R³⁰ is sulfonic acid or carboxylic acid;-   R³¹ and R³⁴ are independently H, F, or Cl-   one of R³² and R³³ is H, F, or Cl, and the other of R³² and R³³ is    -L-R_(x) or -L-S_(c),    wherein L is a covalent linkage of the formula    —S(CH₂)_(a)COO(CH₂)_(b)— or the formula —S(CH₂)_(a)CONH(CH₂)_(b)—    wherein a is an integer between 0 and 10, and b is an integer    between 0 and 10 provided that a and b are not both 0; and R_(x) and    S_(c) are as defined above. Preferably, R_(x), where present, is a    carboxylic acid, an activated ester of a carboxylic acid, a    haloacetamide, a hydrazine, an isothiocyanate, a maleimide group, or    a reactive platinum complex; and S_(c), where present, is an amino    acid, a peptide, a protein, an metal chelating moiety, a nucleoside,    a nucleotide, an oligonucleotide, or a nucleic acid.    Reduced Dyes

Selected dye embodiments are interconvertible with the reduced, ordihydroxanthene, form of the dye. These dihydroxanthene derivativeshaving the general structures:

where J′ is the same as J, defined above, R²⁸ is defined as above, R¹³is H, hydroxy, CN or a C₁-C₆ alkoxy, and R¹⁴ is H, C₁-C₁₈ alkyl, or-L-R_(x), or -L-S_(c). The remaining dye substituents are as definedabove.

In another embodiment of the reduced dyes of the invention, wherein R³⁰is a carboxylic acid, or R²⁸ is a propionic or butyric acid, may existin equilibrium with an isomer that incorporates a spirolactone ring.Similarly, reduced dyes wherein R³⁰ is a sulfonic acid, or R²⁸ is asulfonic acid-substituted ethyl or propyl may exist in equilibrium withan isomer that incorporates a spirosultone ring. Isomers thatincorporate a spirolactone or spirosultone ring are typicallynon-fluorescent until the ring is opened.

For these embodiments, R¹³ taken in combination with R²⁸ forms a5-membered spirolactone ring or a 5-membered spirosultone ring.Alternatively, R¹³ in combination with R³⁰ forms a 5- or 6-memberedspirolactone ring or a 5- or 6-membered spirosultone ring.

The dihydroxanthene and xanthylium versions of the dyes of the inventionare freely interconvertible by well-known oxidation or reductionreagents, as discussed below.

Conjugates of Reactive Dyes

In another embodiment of the invention, the cyanine dyes of theinvention are chemically reactive, and are substituted by at least onegroup -L-R_(x), where R_(x) is the reactive group that is attached tothe dye by a covalent linkage L. R_(x) is a reactive group thatfunctions as the site of attachment for another moiety wherein thereactive group chemically reacts with an appropriate reactive orfunctional group on another substance or moiety. These reactive groupsor reactive precursor are synthesized during the formation of thepresent compounds providing present compounds that can be covalentlyattached to another substance, conjugated substance, facilitated by thereactive group. In this way, compounds incorporating a reactive group(R_(x)) can be covalently attached to a wide variety of biomolecules ornon-biomolecules that contain or are modified to contain functionalgroups with suitable reactivity, resulting in chemical attachment of theconjugated substance (S_(c)), represented by -L-S_(c). In this way, thepresent dye compounds can function as reporter molecules for theconjugated substance. The reactive group and functional group aretypically an electrophile and a nucleophile that can generate a covalentlinkage. Alternatively, the reactive group is a photoactivatable group(a benzophenone, an aryl azide or a diazirine), and becomes chemicallyreactive only after illumination with light of an appropriatewavelength. Typically, the conjugation reaction between the reactivegroup and the substance to be conjugated results in one or more atoms ofthe reactive group R_(x) to be incorporated into a new linkage attachingthe compound of the invention to the conjugated substance Sc. Selectedexamples of functional groups and linkages are shown in Table 2, wherethe reaction of an electrophilic group and a nucleophilic group yields acovalent linkage.

TABLE 2 Examples of some routes to useful covalent linkagesElectrophilic Group Nucleophilic Group Resulting Covalent Linkageactivated esters* amines/anilines carboxamides acrylamides thiolsthioethers acyl azides** amines/anilines carboxamides acyl halidesamines/anilines carboxamides acyl halides alcohols/phenols esters acylnitriles alcohols/phenols esters acyl nitriles amines/anilinescarboxamides aldehydes amines/anilines imines aldehydes or ketoneshydrazines hydrazones aldehydes or ketones hydroxylamines oximes alkylhalides amines/anilines alkyl amines alkyl halides carboxylic acidsesters alkyl halides thiols thioethers alkyl halides alcohols/phenolsethers alkyl sulfonates thiols thioethers alkyl sulfonates carboxylicacids esters alkyl sulfonates alcohols/phenols ethers anhydridesalcohols/phenols esters anhydrides amines/anilines carboxamides arylhalides thiols thiophenols aryl halides amines aryl amines aziridinesthiols thioethers boronates glycols boronate esters carboxylic acidsamines/anilines carboxamides carboxylic acids alcohols esters carboxylicacids hydrazines hydrazides carbodiimides carboxylic acids N-acylureasor anhydrides diazoalkanes carboxylic acids esters epoxides thiolsthioethers haloacetamides thiols thioethers haloplatinate amino platinumcomplex haloplatinate heterocycle platinum complex halotriazinesamines/anilines aminotriazines halotriazines alcohols/phenols triazinylethers imido esters amines/anilines amidines isocyanates amines/anilinesureas isocyanates alcohols/phenols urethanes isothiocyanatesamines/anilines thioureas maleimides thiols thioethers phosphoramiditesalcohols phosphite esters silyl halides alcohols silyl ethers sulfonateesters amines/anilines alkyl amines sulfonate esters thiols thioetherssulfonate esters carboxylic acids esters sulfonate esters alcoholsethers sulfonyl halides amines/anilines sulfonamides sulfonyl halidesphenols/alcohols sulfonate esters *Activated esters, as understood inthe art, generally have the formula —CO, where is a good leaving group(e.g. succinimidyloxy (—OC₄H₄O₂) sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H),-1-oxybenzotriazolyl (—OC₆H₄N₃); or an aryloxy group or aryloxysubstituted one or more times by electron withdrawing substituents suchas nitro, fluoro, chloro, cyano, or trifluoromethyl, or combinationsthereof, used to form activated aryl esters; or a carboxylic acidactivated by a carbodiimide to form an anhydride or mixed anhydride—OCOR^(a) or —OCNR^(a)NHR^(b), where R^(a) and R^(b), which may be thesame or different, are C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, or C₁-C₆alkoxy; or cyclohexyl, 3-dimethylaminopropyl, or N-morpholinoethyl).**Acyl azides can also rearrange to isocyanates

The covalent linkage L binds the reactive group R_(x) or conjugatedsubstance S_(c) to the compound, either directly (L is a single bond) orwith a combination of stable chemical bonds, optionally includingsingle, double, triple or aromatic carbon-carbon bonds, as well ascarbon-nitrogen bonds, nitrogen-nitrogen bonds, carbon-oxygen bonds,carbon-sulfur bonds, phosphorus-oxygen bonds, and phosphorus-nitrogenbonds. L typically includes ether, thioether, carboxamide, sulfonamide,urea, urethane or hydrazine moieties. In one embodiment, the covalentlinkage incorporates a platinum atom, such as described in U.S. Pat. No.5,714,327 (incorporated by reference). Preferred L moieties have 1-20nonhydrogen atoms selected from the group consisting of C, N, O, P, andS; and are composed of any combination of ether, thioether, amine,ester, carboxamide, sulfonamide, hydrazide bonds and aromatic orheteroaromatic bonds. Preferably L is a combination of singlecarbon-carbon bonds and carboxamide or thioether bonds. The longestlinear segment of the linkage L preferably contains 4-10 nonhydrogenatoms, including one or two heteroatoms. Examples of L includesubstituted or unsubstituted polymethylene, arylene, alkylarylene,arylenealkyl, or arylthio. In one embodiment, L contains 1-6 carbonatoms; in another, L is a thioether linkage. In yet another embodiment,L is or incorporates the formula —(CH₂)_(a)(CONH(CH₂)_(b))_(z)—, where ahas any value from 0-5, b has any value from 1-5 and z is 0 or 1.

The -L-R_(x) and -L-S_(c) moieties are bound directly to the fluorophoreat any of R¹, R^(1′), R², R^(2′), R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, R¹⁶, R²⁰,R²¹, R²³, R²⁴, R²⁵, R²⁸, R³⁰, R³¹, R³², R³³, R³⁴, R³⁷, R³⁸, R⁴¹, R⁴²,R⁴³, R⁴⁴, R⁴⁵, or R⁴⁶. In one embodiment, exactly one of R²⁸, R³⁰, R³¹,R³², R³³ and R³⁴ is a -L-R_(x) or -L-S_(c) moiety. In anotherembodiment, one of R³⁷ and R³⁸ is -L-R_(x) or -L-S_(c). In yet anotherembodiment, R²⁸ is -L-R_(x) or -L-S_(c). In yet another embodiment,exactly one of R¹⁵ and R¹⁶ is a -L-R_(x) or -L-S_(c) moiety. In anotherembodiment, one of R¹ and R² is an -L-R_(x) and -L-S_(c). In yet anotherembodiment, one of R³ and R⁴ is an -L-R_(x) or -L-S_(c).

Choice of the reactive group used to attach the fluorophore to thesubstance to be conjugated typically depends on the functional group onthe substance to be conjugated and the type or length of covalentlinkage desired. The types of functional groups typically present on theorganic or inorganic substances include, but are not limited to, amines,thiols, alcohols, phenols, aldehydes, ketones, phosphates, imidazoles,hydrazines, hydroxylamines, disubstituted amines, halides, epoxides,sulfonate esters, purines, pyrimidines, carboxylic acids, or acombination of these groups. A single type of reactive site may beavailable on the substance (typical for polysaccharides), or a varietyof sites may occur (e.g. amines, thiols, alcohols, phenols), as istypical for proteins. A conjugated substance may be conjugated to morethan one fluorophore, which may be the same or different, or to asubstance that is additionally modified by a hapten, such as biotin.Although some selectivity can be obtained by careful control of thereaction conditions, selectivity of labeling is best obtained byselection of an appropriate reactive dye.

Typically, R_(x) will react with an amine, a thiol, an alcohol, analdehyde or a ketone. In one embodiment, R_(x) is an acrylamide, anactivated ester of a carboxylic acid, an acyl azide, an acyl nitrile, analdehyde, an alkyl halide, an amine, an anhydride, an aniline, an arylhalide, an azide, an aziridine, a boronate, a carboxylic acid, adiazoalkane, a haloacetamide, a halotriazine, a hydrazine (includinghydrazides), an imido ester, an isocyanate, an isothiocyanate, amaleimide, a phosphoramidite, a reactive platinum complex, a sulfonylhalide, or a thiol group. By “reactive platinum complex” is meantchemically reactive platinum complexes such as described in U.S. Pat.No. 5,714,327.

Where the reactive group is a photoactivatable group, such as an azide,diazirinyl or azidoaryl derivative, the dye becomes chemically reactiveonly after illumination with light of an appropriate wavelength.

Where R_(x) is a succinimidyl ester of a carboxylic acid, the reactivedye is particularly useful for preparing dye-conjugates of proteins oroligonucleotides. Where R_(x) is a maleimide or haloacetamide thereactive dye is particularly useful for conjugation to thiol-containingsubstances. Where R_(x) is a hydrazide, the reactive dye is particularlyuseful for conjugation to periodate-oxidized carbohydrates andglycoproteins, and in addition is an aldehyde-fixable polar tracer forcell microinjection.

Preferably, R_(x) is a phosphoramidite, a succinimidyl ester of acarboxylic acid, a haloacetamide, a hydrazine, an isothiocyanate, amaleimide group, a perfluorobenzamido, or an azidoperfluorobenzamidogroup. More preferably, R_(x) is a phosphoramidite, a reactive platinumcomplex, or a succinimidyl ester of a carboxylic acid. Where R_(x) is areactive platinum complex, it is typically a haloplatinate.

The reactive dyes of the invention are useful for the preparation of anyconjugated substance that possess a suitable functional group forcovalent attachment of the fluorophore. However, it is appreciated thatcertain conjugated substances may be covalently bonded to the presentdyes wherein a reactive group was not employed to bond together thepresent compound and the biological or non-biological substance. This isparticularly the case, wherein a linker of a single covalent bondattaches the present compound to another chemical moiety, such as ametal chelating moiety.

Examples of particularly useful dye-conjugates include, among others,conjugates of antigens, steroids, vitamins, drugs, haptens, metabolites,toxins, environmental pollutants, amino acids, peptides, proteins,nucleic acids, nucleic acid polymers, carbohydrates, lipids, metalchelating moieties, and non-biological polymers. Alternatively, theseare conjugates of cells, cellular systems, cellular fragments, orsubcellular particles. Examples include, among others, virus particles,bacterial particles, virus components, biological cells (such as animalcells, plant cells, bacteria, yeast, or protists), or cellularcomponents. Reactive dyes typically label reactive sites at the cellsurface, in cell membranes, organelles, or cytoplasm. Preferably theconjugated substance is an amino acid, peptide, protein, tyramine (seeExample 42), polysaccharide, metal chelating moiety, nucleoside,nucleotide, oligonucleotide, nucleic acid, hapten, psoralen, drug,hormone, lipid, lipid assembly, polymer, polymeric microparticle,biological cell or virus. In one embodiment, conjugates of biologicalpolymers such as peptides, proteins, oligonucleotides, nucleic acidpolymers are also labeled with a second fluorescent or non-fluorescentdye, including an additional dye of the present invention, to form anenergy-transfer pair.

In one embodiment, the conjugated substance (S_(c)) is an amino acid(including those that are protected or are substituted by phosphates,carbohydrates, or C₁ to C₂₂ carboxylic acids), or is a polymer of aminoacids such as a peptide or protein. Preferred conjugates of peptidescontain at least five amino acids, more preferably 5 to 36 amino acids.Preferred peptides include, but are not limited to, neuropeptides,cytokines, toxins, protease substrates, and protein kinase substrates.Preferred protein conjugates include enzymes, antibodies, lectins,glycoproteins, histones, albumins, lipoproteins, avidin, streptavidin,protein A, protein G, phycobiliproteins and other fluorescent proteins,hormones, toxins and growth factors. Typically, the conjugated proteinis an antibody, an antibody fragment, avidin, streptavidin, a toxin, alectin, a hormone, or a growth factor. Typically where the conjugatedsubstance is a toxin, it is a neuropeptide or a phallotoxin, such asphalloidin.

In another embodiment, the conjugated substance (S_(c)) is a nucleicacid base, nucleoside, nucleotide or a nucleic acid polymer, includingthose that were modified to possess an additional linker or spacer forattachment of the dyes of the invention, such as an alkynyl linkage(U.S. Pat. No. 5,047,519), an aminoallyl linkage (U.S. Pat. No.4,711,955) or other linkage. In another embodiment, the conjugatedsubstance is a nucleoside or nucleotide analog that links a purine orpyrimidine base to a phosphate or polyphosphate moiety through anoncyclic spacer (acyclonucleosides and acyclonucleotides). Preferably,the conjugated nucleotide is a nucleoside triphosphate or adeoxynucleoside triphosphate or a dideoxynucleoside triphosphate.

Preferred nucleic acid polymer conjugates are labeled, single- ormulti-stranded, natural or synthetic DNA or RNA, DNA or RNAoligonucleotides, or DNA/RNA hybrids, or incorporate an unusual linkersuch as morpholine derivatized phosphates (AntiVirals, Inc., CorvallisOreg.), or peptide nucleic acids such as N-(2-aminoethyl)glycine units.When the nucleic acid is a synthetic oligonucleotide, it typicallycontains fewer than 50 nucleotides, more typically fewer than 25nucleotides.

Large fluorescent nucleic acid polymers are typically prepared fromlabeled nucleotides or oligonucleotides using oligonucleotide-primed DNApolymerization, such as by using the polymerase chain reaction orthrough primer extension, or by terminal-transferase catalyzed additionof a labeled nucleotide to a 3′-end of a nucleic acid polymer.Typically, the dye is attached via one or more purine or pyrimidinebases through an amide, ester, ether or thioether bond; or is attachedto the phosphate or carbohydrate by a bond that is an ester, thioester,amide, ether or thioether. Alternatively, dye conjugate of the inventionis simultaneously labeled with a hapten such as biotin or digoxigenin,or to an enzyme such as alkaline phosphatase, or to a protein such as anantibody. Nucleotide conjugates of the invention are readilyincorporated by DNA polymerase and can be used for in situ hybridization(Example 62) and nucleic acid sequencing (e.g., U.S. Pat. Nos.5,332,666; 5,171,534; and 4,997,928; and WO Appl. 94/05688). In anotheraspect of the invention, the oligonucleotide incorporates an aliphaticamine, which is then conjugated to an amine-reactive dye of theinvention. In yet another aspect of the invention, the purine bases ofthe oligonucleotide react with a reactive platinum complex bound to adye of the invention, yielding a dye-conjugate (Example 65).

In one embodiment, the conjugated oligonucleotides of the invention areaptamers for a particular target molecule, such as a metabolite, dye,hapten, or protein. That is, the oligonucleotides have been selected tobind preferentially to the target molecule. Methods of preparing andscreening aptamers for a given target molecule have been previouslydescribed and are known in the art (for example U.S. Pat. No.5,567,588).

In another embodiment, the conjugated substance (S_(c)) is acarbohydrate that is typically a polysaccharide, such as a dextran,FICOLL, heparin, glycogen, amylopectin, mannan, inulin, starch, agaroseand cellulose. Alternatively, the carbohydrate is a polysaccharide thatis a lipopolysaccharide. Preferred polysaccharide conjugates aredextran, FICOLL, or lipopolysaccharide conjugates.

In another embodiment, the conjugated substance (S_(c)), is a lipid(typically having 6-60 carbons), including glycolipids, phospholipids,sphingolipids, and steroids. Alternatively, the conjugated substance isa lipid assembly, such as a liposome. The lipophilic moiety may be usedto retain the conjugated substances in cells, as described in U.S. Pat.No. 5,208,148. Certain polar dyes of the invention may also be trappedwithin lipid assemblies.

Conjugates having a metal chelating moiety serve as indicators forcalcium, sodium, magnesium, zinc, potassium, gallium, iron, lead orother important metal ions. Preferred metal chelating moieties are crownethers, including diaryldiaza crown ethers (U.S. Pat. No. 5,405,975);derivatives of 1,2-bis-(2-aminophenoxyethane)-N,N,N′,N′-tetraacetic acid(BAPTA chelators; U.S. Pat. Nos. 5,453,517, 5,516,911, and 5,049,673);derivatives of 2-carboxymethoxy-aniline-N,N-diacetic acid (APTRAchelators; AM. J. PHYSIOL. 256, C540 (1989)); or pyridine- andphenanthroline-based metal ion chelators (U.S. Pat. No. 5,648,270).Preferably the metal chelating moiety is a diaryldiaza crown ether, aBAPTA chelator, or an APTRA chelator. These metal chelating moieties aretypically bonded to the present compounds by a single bond wherein areactive group and conjugation reaction was not employed to form thedye-conjugate. Instead these compounds were synthesized utilizing adifferent strategy, See Example 39 (Compound 59), Example 69 (Compound65) and Example 70 (Compound 66).

In one aspect of the invention, these metal chelating moieties that arebonded to the present compounds, are substituted by a reactive group. Ofparticular interest are photoactivatable reactive groups (abenzophenone, an aryl azide or a diazirine) such that a covalent bondcan be formed, after activation with an appropriate light source such asa UV light, with a metal ion or metal ion containing compound that iscomplexed with the metal chelating moiety-dye compound. In thisinstance, a very specific covalent bond can be formed without the needto purify the sample and allows for indirect labeling of the present dyecompounds to a metal ion containing complex. Metal ion containingcompounds include, but are not limited to, zinc-binding proteins,calcium binding proteins and other proteins that bind biological andnon-biological metal ions.

Alternatively, where the dye is a derivative of a rhodol or aseminaphthorhodafluor (SNARF), the dye itself acts as an indicator of H⁺at pH values within about 1.5 pH units of the individual dye's pKa (seeExample 44).

Where the conjugated substance of the -L-S_(c) moiety is a metalchelating moiety, -L-S_(c) is typically R²⁸, or one of R³⁰-R³⁴.Alternatively, the metal chelating moiety is bound at one of R³⁷ or R³⁸.The ion indicators are optionally conjugated to plastic or biologicalpolymers such as dextrans or microspheres to improve their utility assensors.

Other conjugates of non-biological materials include dye-conjugates oforganic or inorganic polymers, polymeric films, polymeric wafers,polymeric membranes, polymeric particles, or polymeric microparticles,including magnetic and non-magnetic microspheres, conducting andnon-conducting metals and non-metals, and glass and plastic surfaces andparticles. Conjugates are optionally prepared by copolymerization of adye that contains an appropriate functionality while preparing thepolymer, or by chemical modification of a polymer that containsfunctional groups with suitable chemical reactivity. Other types ofreactions that are useful for preparing dye-conjugates of polymersinclude catalyzed polymerizations or copolymerizations of alkenes andreactions of dienes with dienophiles, transesterifications ortransaminations. In another embodiment, the conjugated substance is aglass or silica, which may be formed into an optical fiber or otherstructure.

The preparation of dye conjugates using reactive dyes is welldocumented, e.g. by R. Haugland, MOLECULAR PROBES HANDBOOK OFFLUORESCENT PROBES AND RESEARCH CHEMICALS, Chapters 1-3 (1996); andBrinkley, BIOCONJUGATE CHEM., 3, 2 (1992). Conjugates typically resultfrom mixing appropriate reactive dyes and the substance to be conjugatedin a suitable solvent in which both are soluble. The dyes of theinvention are readily soluble in aqueous solutions, facilitatingconjugation reactions with most biological materials.

For those reactive dyes that are photoactivated, conjugation requiresillumination of the reaction mixture to activate the reactive dye.

Labeled members of a specific binding pair are typically used asfluorescent probes for the complementary member of that specific bindingpair, each specific binding pair member having an area on the surface orin a cavity that specifically binds to and is complementary with aparticular spatial and polar organization of the other. Preferredspecific binding pair members are proteins that bind non-covalently tolow molecular weight ligands, such as biotin, drug-haptens andfluorescent dyes (such as an anti-fluorescein antibody). Such probesoptionally contain a covalently bound moiety that is removed by anenzyme or light, or the dye is a dihydroxanthene derivative where R¹³ isH and the compound fluoresces following oxidation. Representativespecific binding pairs are shown in Table 3.

TABLE 3 Representative Specific Binding Pairs antigen antibody biotinavidin (or streptavidin or anti-biotin) IgG* protein A or protein G drugdrug receptor toxin toxin receptor carbohydrate lectin or carbohydratereceptor peptide peptide receptor protein protein receptor enzymesubstrate enzyme DNA (RNA) aDNA (aRNA)† hormone hormone receptor ionchelator psoralen nucleic acid target molecule RNA or DNA aptamer *IgGis an immunoglobulin †aDNA and aRNA are the antisense (complementary)strands used for hybridization

In one aspect of the invention, the conjugated substance is furtherlabeled with additional dye moieties, such that fluorescence energy iseither accepted from, or transferred to, the dye of the invention. Asstated above, N-aryl derivatives of the dyes of the invention haveparticular utility as quenchers in FRET applications. However,fluorescent dyes of the invention also possess utility as energy donorsin FRET applications.

Applications and Methods of Use

In one aspect of the invention, the dye compounds of the inventionpossess utility as laser dyes according to methods known in the art. Asdiscussed above, the long wavelength properties of the subject dyesallow the use of inexpensive laser diodes as excitation sources for dyelasers utilizing the subject dyes.

In another aspect of the invention, the dye compounds of the inventionare used to directly stain or label a sample so that the sample can beidentified or quantitated. For instance, such dyes may be added as partof an assay for a biological target analyte, as a detectable tracerelement in a biological or non-biological fluid, or for such purposes asphotodynamic therapy of tumors, in which a dyed sample is irradiated toselectively destroy tumor cells and tissues, or to photoablate arterialplaque or cells, usually through the photosensitized production ofsinglet oxygen.

In one aspect of the invention, the sample is obtained directly from aliquid source or as a wash from a solid material (organic or inorganic)or a growth medium in which cells have been introduced for culturing, ora buffer solution in which cells have been placed for evaluation. Wherethe sample comprises cells, the cells are optionally single cells,including microorganisms, or multiple cells associated with other cellsin two or three dimensional layers, including multicellular organisms,embryos, tissues, biopsies, filaments, biofilms, etc.

Alternatively, the sample is a solid, optionally a smear or scrape or aretentate removed from a liquid or vapor by filtration. In one aspect ofthe invention, the sample is obtained from a biological fluid, includingseparated or unfiltered biological fluids such as urine, cerebrospinalfluid, blood, lymph fluids, tissue homogenate, interstitial fluid, cellextracts, mucus, saliva, sputum, stool, physiological secretions orother similar fluids. Alternatively, the sample is obtained from anenvironmental source such as soil, water, or air; or from an industrialsource such as taken from a waste stream, a water source, a supply line,or a production lot.

In yet another embodiment, the sample is present on or in solid orsemi-solid matrix. In one aspect of the invention, the matrix is amembrane. In another aspect, the matrix is an electrophoretic gel, suchas is used for separating and characterizing nucleic acids or proteins.In another aspect, the matrix is a silicon chip or glass slide, and theanalyte of interest has been immobilized on the chip or slide in anarray. In yet another aspect, the matrix is a microwell plate ormicrofluidic chip, and the sample is analyzed by automated methods,typically by various methods of high-throughput screening, such as drugscreening.

The dye compounds of the invention are generally utilized by combining adye compound of the invention as described above with the sample ofinterest under conditions selected to yield a detectable opticalresponse. The term “dye compound” is used herein to refer to all aspectsof the claimed dyes, including both reactive and non-reactive dyes andconjugates of dyes. The dye compound typically forms a covalent ornon-covalent association or complex with an element of the sample, or issimply present within the bounds of the sample or portion of the sample.The sample is then illuminated at a wavelength selected to elicit theoptical response. Typically, staining the sample is used to determine aspecified characteristic of the sample by further comparing the opticalresponse with a standard or expected response.

A detectable optical response means a change in, or occurrence of, anoptical signal that is detectable either by observation orinstrumentally. Typically the detectable response is a change influorescence, such as a change in the intensity, excitation or emissionwavelength distribution of fluorescence, fluorescence lifetime,fluorescence polarization, or a combination thereof. The degree and/orlocation of staining, compared with a standard or expected response,indicates whether and to what degree the sample possesses a givencharacteristic. Some dyes of the invention, such as N-aryl rhodaminesand triphenyl methane derivatives, may exhibit little fluorescenceemission, but are still useful as chromophoric dyes. Such chromophoresare useful as energy acceptors in FRET applications, or to simply impartthe desired color to a sample or portion of a sample.

For biological applications, the dye compounds of the invention aretypically used in an aqueous, mostly aqueous or aqueous-misciblesolution prepared according to methods generally known in the art. Theexact concentration of dye compound is dependent upon the experimentalconditions and the desired results, but typically ranges from about onenanomolar to one millimolar or more. The optimal concentration isdetermined by systematic variation until satisfactory results withminimal background fluorescence is accomplished.

The dye compounds are most advantageously used to stain samples withbiological components. The sample may comprise heterogeneous mixtures ofcomponents (including intact cells, cell extracts, bacteria, viruses,organelles, and mixtures thereof), or a single component or homogeneousgroup of components (e.g. natural or synthetic amino acid, nucleic acidor carbohydrate polymers, or lipid membrane complexes). These dyes aregenerally non-toxic to living cells and other biological components,within the concentrations of use.

The dye compound is combined with the sample in any way that facilitatescontact between the dye compound and the sample components of interest.Typically, the dye compound or a solution containing the dye compound issimply added to the sample. Certain dyes of the invention, particularlythose that are substituted by one or more sulfonic acid moieties, tendto be impermeant to membranes of biological cells, and once insideviable cells are typically well retained. Treatments that permeabilizethe plasma membrane, such as electroporation, shock treatments or highextracellular ATP can be used to introduce dye compounds into cells.Alternatively, the dye compounds can be physically inserted into cells,e.g. by pressure microinjection, scrape loading, patch clamp methods, orphagocytosis.

Dyes that incorporate an amine or a hydrazine residue can bemicroinjected into cells, where they can be fixed in place by aldehydefixatives such as formaldehyde or glutaraldehyde. This fixability makessuch dyes useful for intracellular applications such as neuronaltracing.

Dye compounds that possess a lipophilic substituent, such asphospholipids, will non-covalently incorporate into lipid assemblies,e.g. for use as probes for membrane structure; or for incorporation inliposomes, lipoproteins, films, plastics, lipophilic microspheres orsimilar materials; or for tracing. Lipophilic dyes are useful asfluorescent probes of membrane structure.

Chemically reactive dye compounds will covalently attach to acorresponding functional group on a wide variety of materials, formingdye conjugates as described above. Using dye compounds to label reactivesites on the surface of cells, in cell membranes or in intracellularcompartments such as organelles, or in the cell's cytoplasm, permits thedetermination of their presence or quantity, accessibility, or theirspatial and temporal distribution in the sample. Photoreactive dyes canbe used similarly to photolabel components of the outer membrane ofbiological cells or as photo-fixable polar tracers for cells.

Optionally, the sample is washed after staining to remove residual,excess or unbound dye compound. The sample is optionally combined withone or more other solutions in the course of staining, including washsolutions, permeabilization and/or fixation solutions, and solutionscontaining additional detection reagents. An additional detectionreagent typically produces a detectable response due to the presence ofa specific cell component, intracellular substance, or cellularcondition, according to methods generally known in the art. Where theadditional detection reagent has, or yields a product with, spectralproperties that differ from those of the subject dye compounds,multi-color applications are possible. This is particularly useful wherethe additional detection reagent is a dye or dye-conjugate of thepresent invention having spectral properties that are detectablydistinct from those of the staining dye.

The compounds of the invention that are dye conjugates are usedaccording to methods extensively known in the art; e.g. use of antibodyconjugates in microscopy and immunofluorescent assays; and nucleotide oroligonucleotide conjugates for nucleic acid hybridization assays andnucleic acid sequencing (e.g., U.S. Pat. Nos. 5,332,666; 5,171,534;4,997,928; and WO Appl. 94/05688). Dye-conjugates of multipleindependent dyes of the invention possess utility for multi-colorapplications.

At any time after or during staining, the sample is illuminated with awavelength of light selected to give a detectable optical response, andobserved with a means for detecting the optical response. Equipment thatis useful for illuminating the dye compounds of the invention includes,but is not limited to, hand-held ultraviolet lamps, mercury arc lamps,xenon lamps, lasers and laser diodes. These illumination sources areoptionally integrated into laser scanners, fluorescence microplatereaders, standard or minifluorometers, or chromatographic detectors.

The optical response is optionally detected by visual inspection, or byuse of any of the following devices: CCD cameras, video cameras,photographic film, laser-scanning devices, fluorometers, photodiodes,quantum counters, epifluorescence microscopes, scanning microscopes,flow cytometers, fluorescence microplate readers, or by means foramplifying the signal such as photomultiplier tubes. Where the sample isexamined using a flow cytometer, examination of the sample optionallyincludes sorting portions of the sample according to their fluorescenceresponse.

Kits

One aspect of the instant invention is the formulation of kits thatfacilitate the practice of various assays using the dyes of theinvention, as described above. The kits of the invention typicallycomprise a colored or fluorescent dye of the invention, either presentas a chemically reactive label useful for preparing dye-conjugates, orpresent as a dye-conjugate where the conjugated substance is a specificbinding pair member, or a nucleoside, nucleotide, oligonucleotide,nucleic acid polymer, peptide, or protein. The kit optionally furthercomprises one or more buffering agents, typically present as an aqueoussolution. The kits of the invention optionally further compriseadditional detection reagents, a purification medium for purifying theresulting labeled substance, luminescence standards, enzymes, enzymeinhibitors, organic solvent, or instructions for carrying out an assayof the invention.

The examples below are given so as to illustrate the practice of thisinvention. They are not intended to limit or define the entire scope ofthis invention.

A detailed description of the invention having been provided above, thefollowing examples are given for the purpose of illustrating theinvention and shall not be construed as being a limitation on the scopeof the invention or claims.

EXAMPLES Example 1 Preparation of Compound 1

The following compound is prepared:

To a flask (250 mL) is added 4-bromo-3-nitroanisole (57.7 mmol),thiophene-2-boronic acid (69.2 mmol), Pd(OAc)₂ (4.5 mmol), K₂CO₃ (14.3mmol) and Bu₄NBr (115 mmol). The flask is flushed with N₂ gas andequipped with a rubber septum. To the reaction mixture is addeddeoxygenated water (120 mL) with a syringe, and the suspension isstirred and degassed to remove residual oxygen. The suspension isstirred and heated for 1 h at 80° C. under N₂ gas, then cooled to roomtemperature and diluted with water. The resulting precipitate isfiltered and washed with ethyl acetate. The filtrate is extracted withethyl acetate, and the combined organic layers are dried over anhydrousNa₂SO₄ and volatiles are removed under vacuum to give a brown solid. Thecrude solid is purified on a silica gel column eluting with hexanes and5:1 hexanes/ethyl acetate to give Compound 1 (15.2 g).

Compounds 2, 3, and 4 are prepared analogously, usingbenzothiophene-2-boronic acid and 4-bromo-3-nitroanisole,thiophene-2-boronic acid and 2-bromonitrobenzene, andbenzothiophene-2-boronic acid and 2-bromonitrobenzene, respectively.

Example 2 Preparation of Compound 5

The following compound is prepared:

Compound 1 (75 mmol) is dissolved in 1:1 THF/methanol (160 mL). To thesolution is added Zn dust (1.3 moles), followed by the dropwise additionof concentrate hydrochloric acid until analysis by thin layerchromatography (TLC) showed that Compound 1 and the reductionintermediates are completely consumed. Excess Zn is removed byfiltration and washed with THF. The combined filtrates are concentratedin vacuo, and the residue is poured into water (1 L), and neutralized topH=7-8 with 2 M NaOH. The resulting suspension is extracted with ethylacetate. The organic phase is washed with brine, dried over anhydrousNa₂SO₄, and evaporated in vacuo to give the crude amino derivative.

The amino derivative is dissolved in THF (200 mL), and to the resultingsolution is added Ac₂O (20 mL) and pyridine (20 mL) at room temperature.The reaction mixture is stirred at room temperature for 5-6 h, and thenheated at reflux for 1-2 h. The mixture is concentrated to an oil, whichis then redissolved in ethyl acetate (500 mL) and washed with 5% HCl andbrine respectively. The ethyl acetate solution is dried over anhydrousNa₂SO₄, concentrated and loaded on a silica gel column, which is elutedwith a gradient of hexanes/ethyl acetate to give Compound 5 (16.2 g).

Example 3 Preparation of Compound 6

The following compound is prepared:

Compound 5 (65.6 mmol) is put in a dry flask (150 mL). To the flask iscarefully added POCl₃ (80 mL). The mixture is stirred and heated at 80°C. for 30 min. The solution is concentrated in vacuo, and the residue isdiluted with chloroform (500 mL). The chloroform solution is washed withice/water (250 mL) and 5% ammonia (250 mL), then dried over anhydrousNa₂SO₄, and evaporated to afford a brown crude product. The crudeproduct is further purified on a silica gel column using chloroform asthe eluant to yield Compound 6 (10.2 g).

Example 4 Preparation of Compound 7

The following compound is prepared:

To a solution of Compound 6 (44.5 mmol) in chlorobenzene (150 mL), isadded p-TsOMe (237 mmol), and the resulting mixture is heated at refluxfor 2 days. The reaction mixture is then cooled to room temperature, andthe resulting precipitate is collected by filtration. The resultingcrude product is purified by recrystallization from ethyl acetate togive Compound 7 (10.5 g).

Example 5 Preparation of Compound 8

The following compound is prepared:

A solution is prepared of Compound 7 (7.2 mmol) in dry THF (50 mL) at 0°C. under N₂ gas. To the solution is slowly added MeMgCl (30 mmol) inTHF. The reaction mixture is slowly warmed to room temperature andstirred for 2 days. The suspension is carefully poured onto crushed iceand acidified to pH=2 with 2 M HCl. The solution is then extracted withethyl acetate, washed with brine and dried over anhydrous Na₂SO₄. Thesolution is concentrated in vacuo and purified on a silica gel columnusing 5:1 hexanes/ethyl acetate as the eluant to give Compound 8 (600mg).

Example 6 Preparation of Compound 9

The following compound is prepared:

To a solution of Compound 8 (1.25 mmol) in dry CH₂Cl₂ (20 mL) is addedBBr₃ (0.6 mmol) at 0° C., and the mixture is stirred at room temperaturefor 1 h. The reaction mixture is concentrated in vacuo, and cooled in adry ice/acetone bath. To the residue is slowly added methanol (10 mL) toquench the residual BBr₃, followed by the addition of 100 mL water. Thesolution is neutralized to pH=7-8 with saturated NaHCO₃, extracted withethyl acetate and washed with brine. The ethyl acetate solution is driedover anhydrous Na₂SO₄ and concentrated. The resulting residue ispurified on a silica gel column using a gradient of hexanes/ethylacetate as eluant to give Compound 9 (280 mg).

Example 7 Preparation of Compounds 10-12

The following compounds are prepared:

Compound 10 is prepared from Compound 2 following the procedures ofExamples 2-6.

Compound 11 is prepared from furan-2-boronic acid following theprocedures of Examples 1-6.

Compound 12 is prepared from N-methylindole-2-boronic acid following theprocedures of Examples 1-6.

Compound 60 is prepared from thiophene-3-boronic acid following theprocedures of Examples 1-6.

Compound 61 is prepared from benzothiophene-3-boronic acid following theprocedures of Examples 1-6.

Example 8 Preparation of Compound 13

The following compound is prepared:

To a solution of Compound 6 (10 mmol) in chlorobenzene (50 mL), is addedβ-propiolactone (20 mmol). The solution is heated at reflux for 2 days,then cooled to room temperature, and the resulting precipitate collectedby filtration. The crude product is purified by recrystallization fromethyl acetate to give Compound 13 (1.2 g).

Example 9 Preparation of Compound 14

The following compound is prepared:

Compound 13 is treated with MeMgCl to give Compound 14, following theprocedure of Example 5.

Example 10 Preparation of Compound 15

The following compound is prepared:

Compound 14 is treated with BBr₃ to give Compound 15, following theprocedure of Example 6.

Example 11 Preparation of Compound 16

The following compound is prepared:

To a solution of Compound 6 (20 mmol) in chlorobenzene (80 mL), is added1,3-propanesultone (30 mmol), and the reaction mixture is heated atreflux for 3 days. After cooling to room temperature, the reactionmixture is filtered and the precipitate collected. The crude material iswashed with chloroform and recrystallized from isopropyl alcohol to giveCompound 16 (1.4 g).

Example 12 Preparation of Compound 17

The following compound is prepared:

Compound 16 is treated with MeMgCl to give Compound 17, following theprocedure of Example 5.

Example 13 Preparation of Compound 18

The following compound is prepared:

Compound 16 is treated with BBr₃ to give Compound 18, following theprocedure of Example 6.

Example 14 Preparation of Compound 19

The following compound is prepared:

To a solution of Compound 7 (6 mmol) in dry THF (100 mL) is slowly added6-(2,4,10-trioxatricyclo[3.3.1.1]dec-3-yl)hexylmagnesium bromide (50mmol) in THF at 0° C. under N₂ gas. The reaction mixture is slowlywarmed to room temperature, and stirred for 2 days. The suspension iscarefully poured onto crushed ice, and acidified to pH=2 with 2 M HCl.The solution is extracted with ethyl acetate, and evaporated in vacuo todryness. The residue is redissolved in methanol (25 mL), 5 M HCl (10mmol) is added, and the solution is stirred at room temperatureovernight. The reaction mixture is concentrated in vacuo, poured intowater, and extracted with ethyl acetate. The combined organic layers arewashed with brine, dried over anhydrous Na₂SO₄, and evaporated in vacuoto give a crude product, which is further purified by chromatography onsilica gel using 4:1 chloroform/methanol as the eluant to give Compound19 (615 mg).

Example 15 Preparation of Compound 20

The following compound is prepared:

Compound 19 is treated with BBr₃ to give Compound 20, following theprocedure of Example 6.

Example 16 Preparation of Compound 21

The following compound is prepared:

To a solution of Compound 7 (7.2 mmol) in dry THF (50 mL) is slowlyadded PhMgCl (30 mmol) in THF at 0° C. under N₂ gas. The reactionmixture is slowly warmed to room temperature, and stirred for 2 days.The resulting suspension is carefully poured onto crushed ice, acidifiedto pH=2 with 2 M HCl, extracted with ethyl acetate, washed with brineand dried over anhydrous Na₂SO₄. The solution is then concentrated invacuo and the residue is purified by chromatography on silica gel using5:1 hexanes/ethyl acetate as eluant to give Compound 21 (656 mg).

Example 17 Preparation of Compound 22

The following compound is prepared:

Compound 21 is treated with BBr₃ to give Compound 22, following theprocedure of Example 6.

Example 18 Preparation of Compounds 23-36

The following general method is utilized to prepare Compounds 23-36. Aderivatized 3-aminophenol (2 mmol) and a 2-sulfobenzaldehyde precursor(1 mmol) are dissolved in DMF. To the DMF solution is slowly addedtrifluoroacetic acid (TFA, 1 ml) at room temperature. The reactionmixture is stirred overnight, then heated to 110-120° C. for 7-10 h. Themixture is concentrated in vacuo, the residue is dissolved in a minimalamount of chloroform, and purified by chromatography on silica gel toyield the desired product.

TABLE 4 2-sulfobenzaldehyde 3-aminophenol precursor precursor Product

Example 19 Preparation of Compound 37

The following compound is prepared:

POCl₃ (0.3 mL) is dissolved in dry DMF (8 mL) at 0° C., and the mixtureis stirred at room temperature for 0.5 h. To the reaction mixture isadded Compound 10 (2 mmol) in DMF (2 mL). The resulting solution iscarefully poured into cold 10% NaHCO₃, and extracted with ethyl acetate.The combined organic layers are washed with brine and dried overanhydrous Na₂SO₄. The solution is evaporated in vacuo to give Compound37 (516 mg).

Example 20 Preparation of Compound 38

The following compound is prepared:

To a solution of Compound 37 (2 mmol) and dimethylmalonate (2.5 mmol) inmethanol (10 mL) is added piperidine (0.1 mL). The reaction mixture isheated at reflux for 7 h, and concentrated in vacuo. The resultingresidue is poured into water, and extracted with ethyl acetate. Thecombined organic layers are washed with brine and dried over anhydrousNa₂SO₄. The solution is evaporated under vacuum, and the resulting crudematerial is purified by chromatography on silica gel using 50:1chloroform/methanol as the eluant to give Compound 38 (256 mg).

Example 21 Preparation of Compound 39

The following compound is prepared:

A solution of Compound 10 (1 mmol) andN,N-dimethyl-3-hydroxy-4-nitrosoaniline (1.6 mmol) in 1:1butanol/toluene (10 mL) is heated at reflux for 16 h with a Dean-Starktrap. The reaction mixture is concentrated in vacuo, and the residue ispoured into water and extracted with ethyl acetate. The combined organiclayers are washed with brine and dried over anhydrous Na₂SO₄. Theorganic solution is evaporated under vacuum, and the resulting crudematerial is purified by chromatography on silica gel eluting with 5:1chloroform/methanol to give Compound 39 (56 mg).

Example 22 Preparation of Compound 40

The following compound is prepared:

To a solution of Compound 23 (0.14 mmol) in dimethylacetamide (60 mL) isadded thioacetic acid (1.5 mL) at room temperature. The reaction mixtureis heated at 110-120° C. for 2 h, then cooled to room temperature andpoured into water. The suspension is extracted with chloroform, washedwith water, and the chloroform extract is dried over anhydrous Na₂SO₄and concentrated in vacuo. The residue is purified on a silica gelcolumn using 20:1, 10:1, 5:1 chloroform/methanol and 3:1:0.01chloroform/methanol/acetic acid as the eluants to give Compound 40 (90mg).

Compound 62 (Example 18) is treated with thioacetic acid according tothe above procedure to yield the analogous 6-carboxymethylthioderivative, Compound 63.

Example 23 Preparation of Compound 41

The following compound is prepared:

To a solution of Compound 40 (0.12 mmol) in 98% sulfuric acid (5 mL) isslowly added 30% fuming sulfuric acid (4 mL) at 0° C. The reactionmixture is stirred for 10 min at 0° C., and poured into cold ether. Theresulting precipitate is collected, washed with ether, and redissolvedin methanol. The methanol solution is neutralized to pH=7-8 withsaturated Li₂CO₃, and the resulting white precipitate (Li₂SO₄) isremoved by filtration and washed with methanol. The filtrate isconcentrated and purified by HPLC to give Compound 41 (60 mg).

The fluorescence quantum yield of Compound 41 is determined usingsulforhodamine 101 (in ethyl alcohol) as the reference standard(Φ_(F)=0.90). The concentrations of sulforhodamine 101 (the reference)and Compound 41 are adjusted to obtain an absorbance of 0.25 (in 1 cmcell) at 560 nm. The sample and reference are each excited at 560 nm andtheir fluorescence spectra obtained. The fluorescence quantum yield ofthe sample (Φ_(F) ^(X)) in the indicated solvent was calculated from thefollowing formula, considering that the peak area (A_(R)) of theemission spectrum of the reference and that of the tested dye (A_(x))can be readily determined:ΦF^(X)=A_(x)Φ_(F) ^(R)A_(R)where Φ_(F) ^(R) and Φ_(F) ^(X) are the fluorescence quantum yields ofthe reference and the testing dye. The measurements are done intriplicate and the estimated errors are no more than 1%. The quantumyields of Compound 41 are determined to be 0.72 (in methyl alcohol) and0.68 (in water) respectively.

Compound 63 is sulfonated using the above procedure to yield theanalogous disulfonate derivative, Compound 64.

Example 24 Preparation of Compound 42

The following compound is prepared:

To a solution of Compound 41 (0.019 mmol) in dry DMF (10 mL) is addedN,N′ disuccinimidylcarbonate (0.23 mmol) and 4-dimethylaminopyridine(0.04 mmol) at room temperature, and the mixture is stirred for 6 h. Thesolution is then concentrated in vacuo, and the residue is suspended inethyl acetate (100 mL). The precipitate is collected and washed withethyl acetate. The crude solid is redissolved in dry DMF, ethyl acetate(100 mL) is added, and the resulting precipitate is collected, washedwith ethyl acetate, and dried to give Compound 42 (20 mg).

Example 25 Preparation of Compound 43

The following compound is prepared:

To a solution of Compound 42 (0.1 mmol) in anhydrous DMF (2 mL) isslowly added 1 mL DMF solution of N-(5-aminopentyl)maleimide (0.12mmol). To the reaction mixture is then added triethylamine (0.15 mmol),and the resulting mixture is stirred at room temperature for 5-8 h. Thesolution is then concentrated under vacuum and poured into ethylacetate. The resulting precipitate is collected by filtration, andwashed with ethyl acetate. The crude product is purified by HPLC to giveCompound 43 (26 mg).

Example 26 Preparation of Compound 44

The following compound is prepared:

To a solution of hydrazine (0.5 mmol) in DMF (0.05 mL) is slowly added 2mL DMF solution of Compound 42 (0.1 mmol). The resulting mixture isstirred at room temperature for 5-8 h. The reaction solution is thenconcentrated under vacuum and poured into isopropyl alcohol. Theresulting precipitate is collected by filtration, and washed withisopropyl alcohol. The crude material is purified by HPLC to giveCompound 44 (39 mg).

Example 27 Preparation of Compound 45

The following compound is prepared:

Compound 28 (1 mmol) is dissolved in 10:1 methanol/water (60 mL). To theresulting solution is added Na₂S (1.5 moles) and NaSH (4.5 mmol). Thereaction mixture is heated at reflux for 1.5 h, then concentrated invacuo. The residue is poured into water (250 mL) and neutralized topH=7-8 with 2 M HCl. The resulting suspension is extracted withchloroform, the organic phase is washed with brine, dried over anhydrousNa₂SO₄, and evaporated in vacuo. The resulting crude product is furtherpurified by chromatography on silica gel, eluting with a gradient ofchloroform/methanol to give Compound 45.

Example 28 Preparation of Compound 46

The following compound is prepared:

Compound 45 (0.1 mmol) is dissolved in dry DMF (10 mL) and thiophosgene(1 mmol) is added. The reaction mixture is stirred at room temperaturefor 6 h, concentrated in vacuo, and poured into ether. The resultingprecipitate is collected, and washed with ether to give Compound 46 (45mg).

Example 29 Preparation of Compound 47

The following compound is prepared:

Compound 10 (1 mmol), trifluoroacetic anhydride (5 mmol) and TFA (1.5mmol) are dissolved in CH₂Cl₂ (2.5 mL). The reaction mixture is stirredfor 2 days, then concentrated in vacuo, and redissolved in ethanol (1mL) containing 70% perchloric acid (0.1 g). The precipitate iscollected, and purified on a silica gel column using 10:1chloroform/methanol to afford Compound 47 (128 mg).

Example 30 Preparation of Compound 48

The following compound is prepared:

To a solution of Compound 10 (5 mmol) in ethanol (20 mL) is added 37%formaldehyde (20 mmol) at room temperature, and the mixture is stirredat room temperature for 2 days. The resulting yellow solid is collectedand dried. To the solution of the crude solid in concentrated sulfuricacid (5 mL) is slowly added 1 M NaNO₂ at 0° C. The solution is allowedto stand at room temperature for 2 days, and the precipitate iscollected. The crude solid is purified by chromatography on silica gelusing a gradient of chloroform/methanol to give Compound 48 (28 mg).

Example 31 Preparation of Compound 49

The following compound is prepared:

To a solution of Compound 48 (0.5 mmol) in DMF (20 mL) is added KCN (20mmol) at room temperature, and the mixture is stirred at roomtemperature for 2 days. Oxygen gas is bubbled through the solution for 6h. The solution is concentrated in vacuo, and purified by chromatographyon silica gel, eluting with a gradient of chloroform/methanol to giveCompound 49 (31 mg).

Example 32 Preparation of Compound 50

The following compound is prepared:

To a suspension of Compound 10 (1 mmol) and phthalic anhydride (0.4mmol) in propionic acid (10 mL) is slowly added 98% sulfuric acid (0.1mL). The reaction mixture is heated at 130-140° C. for 6 h, thencarefully poured onto crushed ice, and extracted with ethyl acetate. Thecombined organic layers are washed with brine and dried over anhydrousNa₂SO₄. The solution is concentrated in vacuo and purified bychromatography on silica gelusing 5:1 chloroform/methanol as the eluantto give Compound 50 (356 mg).

Example 33 Preparation of Compound 51

The following compound is prepared:

The reaction of Compound 10 (1 mmol) with compoundpyridine-3,4-dicarboxylic anhydride (0.4 mmol) in propionic acid (10 mL)produces Compound 51 analogously with the procedure used to prepareCompound 50.

Example 34 Preparation of Compound 53

The following compound is prepared:

N-methyl-2,2-dimethyl-1,2-dihydrothianaphtheno[3,4]quinoline (Compound52) is prepared from Compound 4 using the procedures of Examples 2-5.Compound 52 (1 mmol) and phthalic anhydride (0.45) are mixed well, andmelted for 15 minutes in vacuo. The mixture is then cooled to roomtemperature, and extracted with methanol (50 mL). The methanol solutionis evaporated under vacuum, and the resulting crude solid is purified bychromatography on silica gel eluting with 10:1:1chloroform/methanol/ethyl acetate to give Compound 53 (156 mg).

Example 35 Preparation of Compound 54

The following compound is prepared:

To a solution of Compound 10 (1 mmol) in methanesulfonic acid (5 mL) isslowly added 2-(2,4-dihydroxybenzoyl)benzoic acid (1.1 mmol). Thereaction mixture is heated at 50-60° C. for 2 h. The solution is thencarefully poured onto crushed ice, and extracted with ethyl acetate. Thecombined organic layers are washed with brine and dried over anhydrousNa₂SO₄. The solution is concentrated in vacuo and purified bychromatography on silica gel using 5:1 chloroform/methanol as the eluantto give Compound 54 (96 mg).

Example 36 Preparation of Compound 55

The following compound is prepared:

To a solution of Compound 10 (1 mmol) and pentafluorobenzaldehyde (0.4mmol) in propionic acid (10 mL) is slowly added p-TsOH (25 mg). Thereaction mixture is heated at 130-140° C. for 6 h, cooled to roomtemperature, and poured into water. The resulting solution is extractedwith ethyl acetate, and the combined organic layers are washed withbrine, dried over anhydrous Na₂SO₄, and evaporated under vacuum. Theresulting crude material is purified by chromatography on silica geleluting with 10:1 chloroform/methanol to give Compound 55 (156 mg).

Example 37 Preparation of Compound 56

The following compound is prepared:

To a solution of Compound 55 (0.1 mmol) and in ethanol (5 mL) is slowlyadded NaBH₄ (5 mmol) at 0° C. The reaction mixture is warmed to roomtemperature, stirred for 2 h, and poured into ice/water. The resultingsuspension is extracted with ethyl acetate, and the combined organiclayers are washed with brine, dried over anhydrous Na₂SO₄, andevaporated under vacuum. The resulting crude material is purified bychromatography on silica gel using 10:1 chloroform/ethyl acetate as theeluant to give Compound 56 (35 mg).

Example 38 Preparation of Compound 57

The following compound is prepared:

To a solution of 2-(2′-carboxybenzoyl)-1,6-dihydroxynaphthalene (1 mmol)in methanesulfonic acid (5 mL) is slowly added Compound 9 (1.1 mmol).The reaction mixture is heated at 50-60° C. for 2 h, the solution iscarefully poured onto crushed ice, and extracted with ethyl acetate. Thecombined organic layers are washed with brine, dried over anhydrousNa₂SO₄, and concentrated under vacuum. The resulting residue is purifiedby chromatography on silica gel using 5:1 chloroform/methanol as eluantto give Compound 57 (76 mg).

Example 39 Preparation of Compound 58

The following compound is prepared:

Compound 10 (0.15 g, 0.51 mmol),1-(2-bis(methoxycarbonylmethyl)amino-5-methyl)-2-(2-bis(methoxycarbonylmethyl)amino-5-formylphenoxy)ethane(0.146 g, 0.25 mmol), and catalytic p-TsOH are suspended in propionicacid (10 mL) under nitrogen atmosphere and sparged with nitrogen for 40minutes while being heated to 60° C. The reaction mixture is heatedovernight. After cooling, the solution is poured into a solution of 11 gsodium acetate in 100 mL water with stirring. The resulting precipitateis collected by suction filtration, rinsed with water, and dried invacuo to give 0.28 g of a pale green powder.

The intermediate (0.28 g; 0.25 mmol) is dissolved in methanol/chloroform(1:1, 12 mL) and treated with p-chloranil (0.123 g, 0.50 mmol). Theresulting mixture is stirred at room temperature until TLC analysisshows consumption of starting material (approximately 1.5 hours). Thereaction mixture is suction filtered and the filtrate concentrated invacuo. The resulting dark blue residue is purified by flashchromatography on 35 g siliga gel using chloroform:methanol:acetic acid(50:5:1) as eluant. Pure product fractions are combined and concentratedin vacuo to give 52 mg of a dark blue solid.

All of the blue intermediate is converted to the free carboxylic acidform by treatment of 52 mg (40 mmol) with 1 M KOH (0.35 mL, 0.35 mmol)in dioxane/methanol (5:3 mL). The resulting dark amber mixture isstirred at room temperature overnight, then concentrated in vacuo. Theresidue is suspended in 5 mL water, and the pH (13) is adjusted to 2 bythe addition of aqueous HCl. The resulting blue precipitate is collectedby filtration, rinsed with water, and dried under vacuum to yield 45 mgof a dark blue powder. The product is further purified dissolution indilute aqueous KOH solution, followed by chromatography on SEPHADEXLH-20 resin using water as eluant. Pure product fractions are combinedand lyophilized to give Compound 58 as a dark blue powder, R_(f)=0.45(dioxane:isopropyl alcohol:water:ammonium hydroxide 15:58:13:13)).

Example 40 Determination of the Ca²⁺ Binding Affinity of Compound 58

The fluorescence response and dissociation constant of Compound 58 isdetermined using the method described by Tsien et al. METH. ENZYM. 172,230 (1989). Compound 58 (1 mg) is dissolved in deionized water and 5 Lof this solution is diluted into 3 mL of each of two buffers, which arecross diluted to arrive at a series of Ca²⁺ concentrations between zeroand 35 M. Emission spectra of the dye solutions are scanned betweendilutions to generate a family of curves. Each of these curves hasmaximal fluorescence emission at approximately 640 nm with an increasein fluorescence emission intensity with increasing Ca²⁺ concentration.This intensity change is plotted against the concentration of free Ca²⁺to give a value for the dissociation constant of the indicator. Thecalculated dissociation constant at 20° C. is 57.5 μM.

Example 41 Preparation of Compound 59

The following compound is prepared:

A light green mixture of 2-carboxymethoxy-4-formyl-aniline-N,N-diaceticacid, trimethyl ester (4-formyl APTRA, trimethyl ester; 60 mg, 0.17mmol), Compound 10 (0.10 g, 0.34 mmol) and catalytic p-TsOH in 8 mLpropionic acid is degassed in vacuo, flushed with nitrogen gas threetimes, then heated to 75 degrees with stirring in darkness overnight.After cooling, the reaction solution is poured into a solution of 10 gsodium acetate in 100 mL water. The resulting precipitate is collectedby suction filtration, rinsed with water, and dried in vacuo to give0.15 g of a green powder (100%, R_(f) 0.30 (EtOAc/hexanes)). All of thispowder (0.17 mmol) is dissolved in 1:1 methanol:chloroform (10 mL), andp-chloranil (50 mg, 0.20 mmol) is added. The resulting mixture isstirred for 3 hours, then concentrated in vacuo. The residue is purifiedby preparative silica gel TLC, using chloroform:methanol:acetic acid(50:5:1) as eluant. The desired product band (R_(f) 0.20) is scrapedfrom the plate. The product is extracted from the silica gel usingchloroform:methanol:acetic acid (50:5:1), followed by filtration andconcentration in vacuo to give 49 mg (31%) of a dark blue powder. Thispowder (40 mg, 0.041 mmol) is dissolved in 4 mL methanol and 1M KOHsolution (0.26 mL, 0.26 mmol) is added. After stirring overnight, TLCshows product formation (R_(f) 0.15 (dioxane:isopropylalcohol:water:ammonium hydroxide 15:58:13:13)) and starting materialconsumption (R_(f) 0.75). The reaction solution is added to 30 mLaqueous citric acid (pH 2). The resulting precipitate is collected on aHirsch funnel, rinsed with water, and dried in vacuo to give Compound 59as 25 mg of a blue powder.

Example 42 Preparation of a Tyramine Conjugate

The following compound is prepared:

To a solution of Compound 42 (0.1 mmol) (Example 25) in anhydrous DMF (2mL) is slowly added 1 mL DMF solution of tyramine (0.22 mmol). Theresulted mixture is stirred at room temperature for 5-8 h until the dyeis completely consumed. The reaction solution is concentrated in vacuo,and poured into ethyl acetate. The resulting precipitate is collected byfiltration and washed with ethyl acetate. The crude material is furtherpurified by HPLC to give the desired product.

Example 43 Labeling of Mitochondria in Live Cells

The NIH/3T3 mouse fibroblast cell line is obtained from American TypeCulture Collection Co., Rockville, Md. The cells are maintained in ahumidified atmosphere of 5% CO₂ in Dulbecco's modified Eagle's medium(DMEM) supplemented with 10% calf serum, 50 μg/mL gentamicin, 300 μg/mLL-glutamine and 10 mM HEPES pH 7.4. Cells are subcultured every 3 daysby trypsinization using 0.05% trypsin and 0.02% EDTA in a Ca- andMg-free saline solution (Gibco BRL, Gaithersburg, Md.). Cell passagenumber ranges from 120-122. To obtain well-spread single cells, 5×10⁴cells are plated onto 18×18 mm coverslips in 60 mm culture dishes.

A cationic dye, such as Compound 48, 49 or 55, is dissolved inDMSO/ethanol (1/1) to prepare a 5 mM stock solution. The stock solutionsare kept sealed in an amber reagent bottle and stored at 4° C. Eachlabeling medium is prepared by adding stock solution to fresh culturemedium in an amount sufficient to make final dye concentrations ofbetween 50 and 200 nM.

The 3T3 cells are transferred to the labeling medium containing theselected dye and incubated at 37° C. for 15 to 30 minutes. The cells arethen washed with fresh medium and observed using a Zeiss Axioplanmicroscope equipped with a filter optimized for tetramethylrhodamine.The selected dyes stain mitochondria selectively and fluorescently.

Example 44 pH Titration of Compound 57

Compound 57 is first dissolved in a series of buffers that have eachbeen calibrated using a pH meter. Acetate buffers are typically used inthe range of pH 4-6, and phosphate buffers in the pH range 6-8.Absorption measurements are made using solutions that are approximately10 M in concentration, and fluorescence measurements are made usingsolutions that are approximately 1 μM in concentration. The absorptionor emission data is then plotted versus pH to determine pKa values. Forexample, FIG. 3 shows the fluorescence emission data for Compound 57plotted versus the pH of the solution, when excited at 338 nm.

Example 45 Preparation of a Phalloidin Dye-Conjugate

To aminophalloidin p-toluenesulfonate (3.5 mg, 4 μmol) and Compound 42(6.0 mg, 5 μmol) in DMF is added N,N-diisopropylethylamine (2 μL, 11μmol). The mixture is stirred at room temperature for 3 hours. To thisis added 7 mL of diethyl ether. The solid is collected bycentrifugation. The crude product is purified on SEPHADEX LH-20, elutingwith water to give the pure phalloidin conjugate.

Example 46 Preparation of a Drug Dye-Conjugate

A fluorescent dopamine D₂ antagonist is prepared as follows: To 10 mg ofN-(p-aminophenethyl)spiperone (Amlaiky et al., FEBS LETT 176, 436(1984)), and 10 μL N,N-diisopropylethylamine in 1 mL of DMF is added 15mg of Compound 42. After 3 hours, the reaction mixture is poured into 5mL ether. The precipitate is centrifuged, then purified bychromatography on silica gel using 10-30% methanol in chloroform.

Example 47 Preparation of Protein Dye-Conjugates

A series of dye conjugates of goat anti-mouse IgG, streptavidin andother proteins, including R-phycoerythrin (R-PE) are prepared bystandard means (Haugland et al., METH. MOL. BIOL. 45, 205 (1995);Haugland, METH. MOL. BIOL. 45, 223 (1995); Haugland, METH. MOL. BIOL.45, 235 (1995)) using Compound 42 and a succinimidyl ester derivative ofCY-5 dye.

A solution of the desired protein is prepared at 10 mg/mL in 0.1 Msodium bicarbonate. The labeling reagents are dissolved in DMF at 10mg/mL. Predetermined amounts of the labeling reagents are added to theprotein solutions with stirring. A molar ratio of 10 equivalents of dyeto 1 equivalent of protein is typical, though the optimal amount varieswith the particular labeling reagent, the protein being labeled and theprotein's concentration, and is determined empirically. The reactionmixture is incubated at room temperature for one hour, or on ice forseveral hours. The dye-protein conjugate is typically separated fromfree unreacted reagent by size-exclusion chromatography on BIO-RAD P-30resin equilibrated with PBS. The initial, protein-containing coloredband is collected and the degree of substitution is determined from theabsorbance at the absorbance maximum of each fluorophore, using theextinction coefficient of the free fluorophore.

TABLE X Fluorescence of Selected Protein Conjugates of the InventionProtein DOS Quantum Yield Goat anti-Mouse IgG 1.2 1.14 Streptavidin 3.600.98 Wheat Germ Agglutinin 1.13 0.56 Conconavilin A 1.10 0.43 Goatanti-Rabbit IgG 1.50 1.36 (highly absorbed) Goat anti-Chicken IgG 1.400.99 Rabbit anti-Mouse IgG 1.30 1.10 Goat anti-Mouse IgG 1.30 1.40(highly absorbed) Goat anti-Guinea Pig IgG 1.20 1.40 Protein A (MR = 4)2.20 1.44 Protein A (MR = 8) 4.40 0.68 Transferrin (MR = 20) 1.30 1.20*Extinction coefficients are determined for the free carboxylic acid inaqueous solution

Protein conjugates of antibody fragments, of other avidins and of otherproteins are prepared and analyzed similarly.

Example 48 Fluorescent Labeling of Periodate-Oxidized Proteins

Two samples of 5 mg each of goat IgG antibody in 1 mL of 0.1 M acetate,0.135 M NaCl, pH 5.5 are treated with 2.1 mg of sodium metaperiodate onice, for 1 and 2 hours, respectively. The reactions are stopped byaddition of 30 μL ethylene glycol. The antibodies are purified on aMATREX GH 25 column (1 cm×30 cm) packed in PBS pH 7.2. One-tenth volumeof 1 M sodium bicarbonate is added to increase the pH and Compound 44 isadded at a molar ratio of dye to protein of 50:1. The reaction isstirred for 2 hours at room temperature. Sodium cyanoborohydride isadded to a final concentration of 10 mM and the reaction is stirred for4 hours at room temperature. The antibody conjugates are purified bydialysis and on MATREX GH 25 columns as described above. Antibodies thatare oxidized for 1 hour typically yield a degree of substitution of 1mole of dye per mole of IgG. Antibodies that are oxidized for 2 hourstypically yield a degree of substitution of approximately 2 mole of dyeper mole of IgG.

Example 49 Total Fluorescence of Selected Dye-Protein Conjugates as aFunction of Degree of Substitution

A series of goat anti-mouse IgG conjugates is prepared as in Example 47so as to yield derivatives with similar degrees of substitution (DOS).The fluorescence emission spectra of a goat anti-mouse IgG conjugate ofCompound 42 (DOS=1.2) and a goat anti-mouse IgG conjugate of CY-5 dye(DOS 2.5) at the same solution optical densities, excited at 600 nm, andin comparison to a solution of DDAO dye having the same absorbance usedas a fluorescence standard. The conjugates of Compound 42 exhibit equalor greater fluorescence than the conjugates of CY-5 dye at similardegrees of substitution.

Example 50 Labeling β-Galactosidase with a Thiol-Reactive Dye

A solution of β-galactosidase, a protein rich in free thiol groups, isprepared in PBS (2.0 mg in 400 μL). The protein solution is then treatedwith a 20 mg/mL solution of Compound 43 in DMF. Unreacted dye is removedon a spin column. The degree of substitution by the dye is estimatedusing the extinction coefficient of the free dye. The proteinconcentration is estimated from the absorbance at 280 nm, corrected forthe absorbance of Compound 43 at that wavelength.

Example 51 Fluorescence Energy Transfer in a Sulfonated-RhodamineConjugate of R-Phycoerythrin

An R-phycoerythrin conjugate, prepared as in Example 47, is excited at488 nm and the fluorescence emission is compared to that of unmodifiedR-phycoerythrin excited at the same wavelength. Highly efficient energytransfer occurs from the protein to the fluorescent dye. A conjugate ofthis complex with streptavidin is prepared essentially as described byHaugland (METH. MOL. BIOL. 45, 205 (1995), supra). This conjugateretains the energy transfer properties and is useful for cell stainingin flow cytometers that utilize the argon-ion laser for excitation.

Example 52 Labeling and Use of a Wheat Germ Agglutinin Dye-Conjugate

Wheat germ agglutinin (100 mg, EY Laboratories) is dissolved in 5 mLNaHCO₃, pH 8.3, containing 9 mg N-acetylglucosamine. To this is added 9mg of Compound 42. After 1 hour the solution is purified by gelfiltration. A degree of substitution of 2-3 dyes per molecule isdetermined from the absorption at 633 nm.

A 1 mg/mL stock solution of the resulting wheat germ agglutinin (WGA)conjugate (Compound 42) is prepared in 0.1 M sodium bicarbonate ˜pH 8.Staphylococcus aureus are cultured for 17 hours at 30° C. in TSB broth.Equal volumes of the TSB culture and a BSA solution (0.25% BSA+0.85%NaCl sterile filtered through 0.2 μM filter) are incubated at roomtemperature for 15 minutes. The BSA-bacterial suspension (200 μL) iscentrifuged for 2 minutes at 350×g, capturing the bacteria on a filtermembrane. The cells are resuspended in 90 μL of BSA solution and 10 μLof stain is added for 15 minutes. Following centrifugation, the bacteriaare resuspended in BSA solution, and an aliquot is trapped between aslide and a glass coverslip.

The bacteria are observed on a Nikon Diaphot epi-fluorescence microscopeusing a fluorescein band pass filter set. Images are acquired using theStar-1 cooled CCD camera and the software package supplied with thecamera is used for data analysis. Two images are collected for eachstain, each image having a 2 sec. exposure time. When used according toSizemore et al. (U.S. Pat. No. 5,137,810) the conjugate can distinguishbetween Gram positive and Gram negative bacteria.

Example 53 Simultaneous Labeling of Actin and Tubulin in CulturedMammalian Cells

Bovine pulmonary artery cells (BPAEC) are grown to 30-50% confluence onglass. The cells are fixed with 3.7% formaldehyde, permeabilized with0.2% Triton X-100, and blocked with 6% bovine serum albumin (BSA). Allcells are incubated with mouse monoclonal anti-α-tubulin for 60 min.

A cell sample is labeled with a monoclonal mouse anti-tubulin (MolecularProbes, Inc.) and a goat anti-mouse IgG conjugate of ALEXA FLUOR 488(Molecular Probes, Inc., Eugene, Oreg.) for 30 min, washed, and thenincubated with the phalloidin dye-conjugate of Example 43 for anadditional 30 min. The cells are rinsed with blocking buffer and mountedin phosphate-buffered saline (PBS) pH 7.4. The stained cells displaymicrotubules decorated with green fluorescence and actin filamentsdecorated with red fluorescence.

Example 54 Utility of Protein Dye-Conjugates as Immunoreagents andResistance to Photobleaching

Goat anti-Mouse IgG conjugates of Compound 42 are prepared with degreesof substitution of approximately 2-4 (as in Example 47). CY5-labelledgoat anti-mouse IgG is purchased from Jackson Immunoresearch. HEp-2 cellslides from INOVA (San Diego, Calif.) are hydrated in 1% bovine serumalbumin (BSA) in PBS for 30 minutes. The slide is drained, humananti-nuclear antibody is applied, the slide is incubated 30 min andrinsed in PBS. Mouse anti-human antibody is applied, the slide isincubated 30 min and rinsed in PBS. The fluorescent anti-mouse antibodyconjugate of choice is applied as a 5 μg/mL solution, diluted in 1%BSA/PBS. After 30 minutes the slides are rinsed in PBS, then in 50 mMTris pH 8.0, mounted in 50 mM Tris pH 8.0, and viewed through anappropriate filter. All samples give predominantly nuclear staining.Quantitative intensity measurements permit comparison of dyes. Similarresults are obtained using a biotinylated anti-mouse preparation andfluorescent streptavidin conjugates.

For photobleaching measurements, one image of the slide is acquiredevery 5 seconds for 100 seconds with continuous illumination. Threefields of cells are bleached, and the photobleaching values arenormalized and averaged. The antibody conjugates of Compound 42 aresignificantly more photostable than those of CY-5 dye (FIG. 2).

Example 55 Preparation and Use of a Fluorescent α-BungarotoxinDye-Conjugate

α-Bungarotoxin (1 mg) in 25 μL 0.1 M NaHCO₃ is treated with 1.5equivalents of Compound 42 at room temperature for 2 hours. The productis purified by size exclusion, by ion exchange chromatography, andfinally by reverse phase HPLC. Staining of acetylcholine receptors anddetection of their resulting fluorescence is comparable to that obtainedwith TEXAS RED dye-conjugated α-bungarotoxin.

Example 56 Preparation of Aminodextran Dye-Conjugates

70,000 MW aminodextran (50 mg) derivatized with an average of 13 aminogroups, is dissolved at 10 mg/mL in 0.1 M NaHCO₃. Compound 46 is addedso as to give dye/dextran ratio of ˜12. After 6 hours the conjugate ispurified on SEPHADEX G-50, eluting with water. Typically 4-6 moles ofdye are conjugated to 70,000 g dextran.

Example 57 Preparation of Fluorescent-Dye Labeled Microspheres

Uniform microspheres are conjugated to the dyes of the invention by oneof five methods. In Method A, 1.0 μm amine-derivatized polystyrenemicrospheres are suspended at ˜2% solids in 100 mM NaHCO₃, pH 8.3 andtreated with 2 mg/mL of an amine-reactive dye. After 1 hour themicrospheres are centrifuged and washed with buffer.

In Method B, carboxylate-modified microspheres are suspended in asolution of a protein that has been conjugated to a dye of theinvention. The protein is passively adsorbed on the microspheres, andexcess protein is removed by centrifugation and washing. Microparticlesof a size that cannot be centrifuged are separated from excess proteinby dialysis through a semi-permeable membrane with a high MW cutoff orby gel filtration chromatography.

In Method C the protein is covalently coupled through its amine residuesto the carboxylate groups of the polymer using ethyl3-(dimethylaminopropyl)carbodiimide (EDAC).

In Method D, biotinylated microspheres are treated with a streptavidin,avidin or anti-biotin conjugate of a dye of the invention, and theconjugates are isolated as in Method B.

In Method E, the microparticle is placed in a nonpolar solution of thedesired dye. The microparticle swells in the presence of the organicsolvent, and the dye is able to permeate the interior of themicroparticle. Upon removing the solvent, the microparticle returns toits normal size, trapping the dye within the microparticle

For example, to a stirred 100 mL suspension of carboxyate-modified latex(Interfacial Dynamics Corp., Portland, Oreg.) that is 4.2% solids isadded 50 mL methanol. A dye solution is prepared that is 25-50 mgdesired dye(s), 7.5 mL methylene chloride and 17.5 mL ethanol. The dyesolution is added to the latex suspension at a low flow rate (˜6 mL/hr),with stirring, using a syringe pump fitted with a Teflon delivery tube.After addition is complete, the organic solvents are removed underreduced pressure, and the aqueous suspension of dyed latex is filteredthrough glass wool to remove any additional debris. The microparticlesare dialyzed in E-pure water (25 mm tubing, MW cutoff 12,000-14,000)until no more free dye is removed from the particles. The fluorescentlatex suspension is filtered again through glass wool and then sonicatedin a bath sonicator for 5 minutes to ensure monodispersity.

The larger particles can be analyzed for uniformity of staining andbrightness using flow cytometry. The microspheres can be further coupledto proteins, oligonucleotides, haptens and other biomolecules for assaysusing methods well know in the art.

Example 58 Preparation of Fluorescent Liposomes Using the Dyes of theInvention

Selected dyes of the invention are sufficiently water soluble to beincorporated into the interior of liposomes by methods well known in theart (J. BIOL. CHEM. 257, 13892 (1982) and PROC. NATL. ACAD. SCI. USA 75,4194 (1978)). Alternatively, liposomes containing dyes of the inventionhaving a lipophilic substituent (e.g. alkyl having 11-22 carbons),within their membranes are prepared by co-dissolving the fluorescentlipid and the unlabeled phospholipid(s) that make up the liposome beforeforming the liposome dispersion essentially as described by Szoka, Jr.et al. (ANN. REV. BIOPHYS. BIOENG. 9, 467 (1980)).

Example 59 Preparation of Fluorescent Dye-Conjugates of Bacteria

Heat-killed Escherichia coli are suspended at 10 mg/mL in pH 8-9 bufferthen incubated with 0.5-1.0 mg/mL of an amine-reactive dye, such asCompound 42. After 30-60 minutes the labeled bacteria are centrifugedand washed several times with buffer to remove any unconjugated dye.Labeled bacteria that are opsonized are taken up by macrophage, asdetermined by flow cytometry.

Example 60 Preparation of a Nucleotide Dye-Conjugate

To 2 mg of 5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate (SigmaChemical) in 100 μL water is added Compound 42 in 100 μL DMF and 5 μLtriethylamine. After 3 hours, the solution is evaporated and the residueis purified by HPLC. The product fractions are lyophilized to give thered fluorescent nucleotide conjugate.

Alternatively fluorescent dye-conjugates of deoxyuridine 5-triphosphateare prepared from 5-(3-amino-1-propynyl)-2′-deoxyuridine 5′-triphosphate(as described in Hobbs, Jr. et al, supra).

Example 61 Preparation of an Oligonucleotide Dye-Conjugate

A 5′-amine-modified, 18-base M13 primer sequence (˜100 μg) is dissolvedin 4 μL 10 mM Tris-HCl, pH 8, 1 mM EDTA. To this is added 250 μg ofCompound 42 in 100 μL 0.1 M sodium borate, pH 8.5. After 16 hours, 10 μLof 5 M NaCl and 3 volumes of cold ethanol are added. The mixture iscooled to −20° C., centrifuged, the supernatant is decanted, the pelletis rinsed with ethanol and then dissolved in 100 μL H₂O. The labeledoligonucleotide is purified by HPLC on a 300A C8 reverse-phase columnusing a ramp gradient of 0.1 M triethylammonium acetate (pH ˜7) andacetonitrile (5→95% over 30 min). The desired peak is collected andevaporated to give the fluorescent oligonucleotide.

Example 62 Preparing DNA Hybridization Probes Using FluorescentNucleotide Dye-Conjugates

For each labeling reaction, a microfuge tube containing about 1 μg of a˜700 bp Hind III-Bgl II fragment of the E. coli lacZ structural gene isheated for ˜10 minutes at 95 C to fully separate the strands. The DNA iscooled on ice. A 2 μL of a 2 mg/mL mixture of random sequencehexanucleotides in 0.5 M Tris-HCl, pH 7.2, 0.1 M MgCl₂, 1 mMdithiothreitol is added, followed by 2 μL of a dNTP labeling mixture (1mM dATP, 1 mM dGTP, 1 mM dCTP, 0.65 mM dTTP and 0.35 mMfluorescent-labeled dUTP (as prepared in Example 60). Sterile distilled,deionized water is added to bring the total volume to 19 L. 1 μL KlenowDNA polymerase (2 units/μL) is added. The samples are incubated 1 hr at37° C. The reactions are stopped with 2 μL of 0.2 M EDTA, pH 8.0. Thelabeled DNA is precipitated with 2.5 μL of 4 M LiCl and 75 μL of −20° C.ethanol. After 2 hours at −20° C. the precipitated nucleic acids arecentrifuged at 12,000 rpm. The pellets are washed with cold 70% ethanol,then cold 100% ethanol. The pellets are dried and dissolved in 10 mMTris-HCl, pH 8.0, 1 mM EDTA. A portion of each sample is analyzed by gelelectrophoresis on a 1% agarose minigel under standard conditions. Thelabeled DNA products are suitable for in situ hybridization experimentsfor the detection of RNA or DNA, such as is associated with the E. colilacZ gene in cells or tissues.

Example 63 Incorporation of Fluorescent Nucleotide Conjugates into DNAAmplification Products

A DNA amplification reaction is prepared as follows: 1 μL each of 20 μMsolutions of two oligonucleotide primers that hybridize to thehuman-actin gene are added to a labeling reaction containing 5 μL DNAtemplate (100 μmol of a plasmid containing the entire gene), 5 μL 10×reaction buffer (100 mM Tris, pH 8.3, 500 mM KCl), 2.5 μL 1 mMfluorescent-labeled dUTP (as prepared in Example 60), 1 μL 10 mM dATP, 1μL 10 mM dCTP, 1 μL 10 mM dGTP, 1.5 μL 5 mM dTTP, 3 μL 25 mM MgCl₂, and28 μL distilled, deionized water. The sample is transferred to athermocycler and processed as follows: one cycle, 94° C., 2.5 minutes;30 cycles, 94° C., 1 minute, 50° C., 1 minute, 72° C., 1 minute; onecycle, 72° C., 5 minutes; then 4° C. overnight. An aliquot of the sampleis mixed with an equal volume of 10% glycerol, loaded onto a 0.9%agarose minigel and electrophoresed. Fluorescent bands of the expectedsize are visible when the gel is illuminated with 300-nm ultravioletlight.

Example 64 In Situ Hybridization of an RNA Probe

Mouse fibroblasts are fixed and prepared for mRNA in situ hybridizationusing standard procedures. A dye-labeled RNA probe is prepared by invitro transcription of a plasmid containing the mouse actin structuralgene cloned downstream of a phage T3 RNA polymerase promoter. Labelingreactions consist of combining 2 μL DNA template (1 μg DNA), 1 μL eachof 10 mM ATP, CTP and GTP, 0.75 μL 10 mM UTP, 2.5 μL 1 mMfluorescent-labeled UTP, 2 μL 10× transcription buffer (400 mM Tris, pH8.0, 100 mM MgCl₂, 20 mM spermidine, 100 mM NaCl), 1 μL T3 RNApolymerase (40 units/μL), 1 μL 2 mg/mL BSA, and 8.75 μL water. Reactionsare incubated at 37° C. for two hours.

The DNA template is removed by treatment with 20 units DNase I for 15minutes, at 37° C. The RNA transcript is purified by extraction with anequal volume of phenol:chloroform, 1:1, then by chromatography onSEPHADEX G50. Labeled RNA is denatured for 5 minutes at 50° C., thenhybridized to cellular preparations using standard procedures. Whenpreparations are washed and viewed through an appropriate filter set ona fluorescence microscope (excitation 610±10 nm; emission 670±20 nm),cells expressing actin mRNA show bright red fluorescence.

Example 65 Preparing DNA Hybridization Probes Using Fluorescent PlatinumDye-Compounds

A fluorescent platinum complex is prepared from a compound of theinvention by adapting the methods provided in U.S. Pat. No. 5,714,327.For each labeling reaction, a microfuge tube containing 1 μg of pUC1.77plasmid DNA containing a chromosome 1 human α-satellite probe (DNasetreated to a fragment size between 500-1000 bp) in 5 mM Tris, pH 8, 1 mMEDTA, is heated for ˜10 minutes at 95° C. to fully denature the DNA. TheDNA is cooled on ice. 1 μL of a 1 mg/mL solution of the preparedplatinum complex is added, followed by the addition of 5 mM Tris, pH 8,1 mM EDTA to bring the total volume to 25 μL. The samples are incubated15 minutes at 80° C. The reactions are stopped on ice. The labeled DNAis purified on a Bio-Rad Micro Bio-Spin P-30 Tris Chromatography Column.The labeled DNA products are suitable for in situ hybridizationexperiments.

Example 66 Preparing DNA Hybridization Probes Using Amine-Modified DNAan Amine-Reactive Dye of the Invention

Nick translation is performed using pUC1.77 plasmid DNA containing achromosome 1 human α-satellite probe. To a microcentrifuge tube isadded, in the following order: 23.5 μL H₂O, 5 μL 10× Nick Translationbuffer (0.5 M Tris-HCL, 50 mM MgCl₂, 0.5 mg/ml BSA, pH 7.8), 5 μL 0.1 MDTT, 4 μL d(GAC)TP mix (0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP), 1 μL 0.5mM dTTP, 4 μL 0.5 mM aminoallyl-dUTP, 1 μL 1 μg/μL template DNA, 5 μLDNase I (1 μg/mL, 2000 Kunitz units/mg), 1.5 μL DNA polymerase I (10U/μL). The tube is incubated 2 hours at 15° C., then brought to a finalvolume of 100 μL with H₂O. The amine-modified DNA is purified using aQIAQUICK PCR purification Kit (Qiagen) with the following modificationsto purify the DNA from the enzyme and amine-containing compounds: 75%EtOH is substituted for the wash buffer, H₂O is substituted for theelution buffer, and elution is performed twice for 5 minutes each. TheDNA is precipitated by adding 1/10 volume 3M sodium acetate and 2.5volumes 100% EtOH, incubated at −70° C. for 30 minutes, centrifuged for15 minutes, and washed with 70% EtOH.

The amine-modified DNA is resuspended in 5 μL H₂O. To the solution isadded 3 μL 25 mg/ml sodium bicarbonate and 50 μg Compound 42 in 5 μLDMF. The reaction is incubated for 1 hour at room temperature in thedark. 90 μL H₂O is added to the reaction and it is purified using aQIAQUICK PCR purification kit (QIAGEN), with the followingmodifications: three washes are performed with 75% EtOH and threeelutions of 5 minutes each with the QIAGEN elution buffer. The DNA isprecipitated as before. The labeled DNA products are suitable for insitu hybridization experiments.

Example 67 Discrimination of Live and Dead Cells Using the Dyes of theInvention

Selected dyes of the invention are highly polar, and thereforerelatively impermeable to the membranes of live cells. These dyes cantherefore be used to discriminate cells that have intact versuscompromised cell membranes in a single-color assay as follows:

Mouse monocyte-macrophage, Abelson Leukemia Virus Transformed (RAW264.7)cells are trypsinized and washed with phosphate buffered saline (PBS),pH 7.2. Approximately 8-10 million cells suspended in 180 μL of PBS, pH7.2 are placed in a glass test tube and heated in a water bath at 50° C.for 20 minutes to kill a fraction of the cells. Approximately 60 μL (2-3million cells) of the cell suspension is added to 940 μL of PBS, pH 7.2,followed by 0.1 μL of a 1 mg/mL solution of Compound 42 in DMSO. Themixture is incubated on ice for 30 minutes and washed twice with PBS,followed by addition of 200 μL of PBS, pH 7.2. An identical aliquot ofcells is treated with 2 μL of a 150 μM solution of propidium iodide inwater (as a control for dead cells). Analysis of the cell suspensionusing flow cytometry shows that populations of dead cells stained byCompound 42 and those stained by propidium iodide are very similar.

Example 68 Neuronal Tracing Using a Hydrazide-Labeled Fluorophore

Neurons from zebrafish embryos are microinjected with Compound 44, usingstandard methods as described by Blankenfeld et al. (J. NEUROSCI. METH.36, 309 (1991)). The neurons rapidly fill with the dye throughout theirvolume and their red fluorescence is readily observable. The staining isfixable in the cells using formaldehyde and standard fixing methods.

Example 69 Preparation of Compound 65

Compound 10 (0.15 g, 0.51 mmol),1-(2-bis(methoxycarbonylmethyl)amino-5-fluoro)-2-(2-bis(methoxycarbonylmethyl)amino-5-formylphenoxy)ethane(0.15 g, 0.25 mmol), and catalytic p-TsOH are suspended in propionicacid (10 mL) under an argon atmosphere and sparged with argon for 40minutes while being heated to 60° C. The reaction mixture is stirred for3 hours, then cooled. The solution is poured into a solution of 3Msodium acetate (100 mL) with stirring. The resulting precipitate iscollected by suction filtration, rinsed with water, and dried in vacuoto give a dihydro intermediate as pale green powder.

The dihydro intermediate (0.25 mmol) is dissolved in methanol/chloroform(1:1, 12 mL) and treated with p-chloranil (0.123 g, 0.50 mmol). Theresulting mixture is stirred at room temperature until TLC analysisshows consumption of starting material (approximately 1.5 hours). Thereaction mixture is suction filtered and the filtrate concentrated invacuo. The resulting dark blue residue is purified by flashchromatography on 35 g silica gel using chloroform:methanol:acetic acid(50:5:1) as eluant. Pure product fractions are combined and concentratedin vacuo to give the oxidized intermediate as a dark blue solid.

The blue oxidized intermediate (0.04 mmol) is converted to the freecarboxylic acid form by treatment with 1 M KOH (0.35 mL, 0.35 mmol) indioxane/methanol (1:1, 5 mL). The resulting dark amber mixture isstirred at room temperature overnight, then concentrated in vacuo. Theresidue is suspended in 5 mL water, and the pH (13) is adjusted to 2 bythe addition of aqueous HCl. The resulting blue precipitate is collectedby filtration, rinsed with water, and dried under vacuum to yield a darkblue powder. The product is further purified by dissolution in diluteaqueous KOH solution, followed by chromatography on SEPHADEX LH-20 resinusing water as eluant. Pure product fractions are combined andlyophilized to give Compound 65 as a dark blue powder, R_(f)=0.45(dioxane:isopropyl alcohol:water:ammonium hydroxide 15:58:13:13)).

Example 70 Preparation of Compound 66

Compound 10 (0.15 g, 0.51 mmol),N,N,N′,N′-tetramethoxycarbonylmethyl-2′-amino-4′-diphenylmethoxycarbonylphenoxy)-2-(2″-aminophenoxy)ethane(0.25 mmol), and catalytic p-TsOH are suspended in propionic acid (10mL) under an argon atmosphere and sparged with argon for 40 minuteswhile being heated to 70° C. The reaction mixture is stirred for 20hours, then cooled. The solution is poured into a solution of 3M sodiumacetate (100 mL) with stirring. The resulting precipitate is collectedby suction filtration, rinsed with water, and dried in vacuo to give adihydro intermediate 4-benzhydryl ester as pale green powder.

The dihydro intermediate (0.25 mmol) is dissolved in methanol/chloroform(1:1, 12 mL) and treated with p-chloranil (0.123 g, 0.50 mmol). Theresulting mixture is stirred at room temperature until TLC analysisshows consumption of starting material (approximately 1.5 hours). Thereaction mixture is suction filtered and the filtrate concentrated invacuo. The resulting dark blue residue is purified by flashchromatography on 35 g silica gel using chloroform:methanol:acetic acid(50:5:1) as eluant. Pure product fractions are combined and concentratedin vacuo to give the oxidized intermediate benzhydryl ester as a darkblue solid.

The oxidized intermediate benzhydryl ester (0.15 mmol) is treated with1:1 chloroform/trifluoroacetic acid (10 mL) for one hour, thenconcentrated in vacuo. Chloroform (2×10 mL) is evaporated from theresidue, which is triturated with ether (20 ml). The resulting oxidizedintermediate 4-carboxylic acid is collected by suction filtration as ablue powder.

The oxidized intermediate carboxylic acid (0.10 mmol) in DMF (2 mL) istreated with diisopropylethylamine (DIEA, 0.35 mL, 2 mmol) and drytrifluoroacetyl-N-hydroxysuccinimide (TFA-SE, 225 mg, 1 mmol). Theresulting blue mixture is stirred for 2 h, then more TFA-SE (113 mg, 0.5mmol) is introduced and the mixture stirred for another 16 h. Themixture is diluted with CHCl₃ (50 mL), washed with 1% AcOH (3×20 mL),H₂O (25 mL), sat. NaCl (50 mL), filtered and evaporated. Ether (25 mL)is added to the residue, and the precipitated product filtered andwashed with ether to give the oxidized intermediate 4-succinimidyl esteras a dark blue solid.

To a solution of 4-aminomethylbenzophenone (0.080 mmol) in DMF (1 mL)and DIEA (0.055 mL, 0.40 mmol) is added a solution of the oxidizedintermediate 4-succinimidyl ester (0.040 mmol) in 1 mL DMF. Theresulting mixture is stirred under subdued light for 3 h, diluted withCHCl₃ (200 mL), washed with 1% AcOH (3×150 mL), H₂O (100 mL), sat. NaCl(200 mL), filtered and evaporated. The residue is purified on twopreparative TLC SiO₂ plates, using 12% MeOH and 2.5% AcOH in CHCl₃ aseluent to give the photoactivatable tetramethyl ester intermediate as adark blue solid.

To a solution of the photoactivatable tetramethyl ester intermediate(0.025 mmol) in MeOH (2 mL) and H₂O (1 mL) is added 1N KOH to give pH12.0. The mixture is stirred under subdued light for 20 h, thenpH-adjusted to 8.5 with 0.1 N HCl. The mixture is evaporated and theresidue purified on a Sephadex LH-20 column (1.6×40 cm bed) using H₂O aseluant. Pure product fractions are pooled and lyophilized to givephotoactivatable Compound 66 as a fluffy dark blue powder.

1. A compound of the formula:

wherein R¹ and R⁴¹ are independently hydrogen or sulfonic acid; R² andR⁴² are independently selected from the group consisting of hydrogen,cyano, halogen, carboxylic acid, sulfonic acid, C₁-C₆ alkyl or alkoxy,aryl, heteroaryl, -L-R, and -L-S_(c), wherein said alkyl or alkoxy isoptionally substituted by carboxylic acid, sulfonic acid, or halogen andsaid aryl or heteroaryl is optionally substituted one or more times byC₁-C₆ alkyl, C₁-C₆ perfluoroalkyl, cyano, halogen, azido, carboxylicacid, sulfonic acid, or halomethyl; R³, R⁴, R⁴³, and R⁴⁴ are eachmethyl; R⁵ and R⁴⁵ are independently selected from the group consistingof hydrogen, methyl, carboxymethyl, C₁-C₆ alkyl, aryl, heteroaryl,-L-R_(x) and -L-S_(c), wherein said alkyl is optionally substituted bycarboxylic acid, sulfonic acid, amino, or halogen and said aryl orheteroaryl is optionally substituted one or more times by C₁-C₆ alkyl,C₁-C₆ perfluoroalkyl, cyano, halogen, carboxylic acid, sulfonic acid, orhalomethyl; R⁶ and R⁴⁶ are both hydrogen; one of E and X is 0, S, orNR⁸; the other is absent; and one of E′ and X′ is O, S, or NR⁸; theother is absent; wherein R⁸ is independently selected from the groupconsisting of hydrogen, methyl, carboxymethyl, C₂-C₆ alkyl, -L-R_(x) and-L-S_(c) wherein said alkyl is optionally substituted by carboxylicacid, sulfonic acid, amino, or halogen; and Q is CR²⁸, wherein R²⁸ is ofthe formula

wherein R³⁰, R³¹, R³², R³³, and R³⁴ are independently selected from thegroup consisting of hydrogen, F, Cl, Br, I, sulfonic acid, carboxylicacid, CN, nitro, hydroxy, azido, amino, hydrazino, C₁-C₁₈ alkyl, C₁-C₁₈alkoxy, C₁-C₁₈ alkylthio, C₁-C₁₈ alkanoylamino, C₁-C₁₈alkylaminocarbonyl, C₂-C₃₆ dialkylaminocarbonyl, C₁-C₁₈alkyloxycarbonyl, C₇-C₁₈ arylcarboxamido, -L-R_(x) and -L-S_(c), whereinsaid alkyl or aryl portions of said R³⁰, R³¹, R³², R³³, and R³⁴ areoptionally substituted one or more times by substituents selected fromthe group consisting of F, Cl, Br, I, hydroxy, carboxylic acid, acarboxylic acid ester of a C₁-C₆ alcohol, amino, C₁-C₆ alkylamino, C₁-C₆dialkylamino and C₁-C₆ alkoxy; or a pair of adjacent R³¹, R³², R³³, andR³⁴ substituents, when taken in combination, form a fused 6-memberedaromatic ring that is optionally further substituted by carboxylic acid;and wherein L is a covalent linkage; R_(x) is a reactive group; andS_(c) is a conjugated substance.
 2. The compound according to claim 1,wherein said L is independently a single covalent bond or a covalentlinkage having 1-20 nonhydrogen atoms selected from the group consistingof C, N, O, P, and S.
 3. The compound according to claim 1, wherein saidR_(x) is independently selected from the group consisting of anacrylamide, an activated ester of a carboxylic acid, an acyl azide, anacyl nitrile, an aldehyde, an alkyl halide, an amine, an anhydride, ananiline, an aryl halide, an azide, an aziridine, a boronate, acarboxylic acid, a diazoalkane, a haloacetamide, a halotriazine, ahydrazine, an imido ester, an isocyanate, an isothiocyanate, amaleimide, a phosphoramidite, a reactive platinum complex, a sulfonylhalide, and a thiol group.
 4. The compound according to claim 1, whereinsaid S_(c) is independently selected from the group consisting of anamino acid, a peptide, a protein, a tyramine, a carbohydrate, a metalchelating moiety, a nucleoside, a nucleotide, an oligonucleotide, anucleic acid, a hapten, a psoralen, a drug, a hormone, a lipid, a lipidassembly, a polymer, a polymeric microparticle, a biological cell, and avirus.
 5. The compound according to claim 1, wherein one of R³⁰, R³¹,R³², R³³, and R³⁴ is -L-R_(x) or -L-S_(c),.
 6. The compound according toclaim 1, of the formula:

wherein R⁵ and R⁴⁵ are independently methyl or ethyl; R³⁰ is sulfonicacid or carboxylic acid; R³¹ and R³⁴ are independently hydrogen, F, orCl; one of R³² and R³³ is H, F, or Cl, and the other of R³² and R³³ is-L-R_(x) or -L-S_(c), wherein L is a covalent linkage of the formula—S(CH₂)_(a)COO(CH₂)_(b)— or the formula S(CH₂)_(a)CONH(CH₂)_(b)— ,wherein a is an integer between 0 and 10, and b is an integer between 0and 10 provided that a and b are not both 0; wherein R_(x), wherepresent, is a carboxylic acid, an activated ester of a carboxylic acid,a haloacetamide, a hydrazine, an isothiocyanate, a maleimide group, or areactive platinum complex; and wherein S_(c), where present, is an aminoacid, a peptide, a protein, an ion-complexing moiety, a nucleoside, anucleotide, an oligonucleotide, lectin or a nucleic acid.
 7. Thecompound according to claim 6, wherein R_(x), is independently amaleimide group or a succinimidyl ester of a carboxylic acid.
 8. Thecompound according to claim 6, wherein S_(c) is independently anantibody or antibody fragment or a BAPTA or APTRA ion-complexing moiety.9. A method of staining a biological sample, comprising: combining a dyesolution comprising a compound of the formula as described in claim 1with a biological sample in a concentration sufficient to yield adetectable optical response under desired conditions.
 10. The methodaccording to claim 9, further comprising: combining the sample with adetection reagent that has spectral properties that are detectablydifferent from said optical response; and/or determining acharacteristic of the sample by comparing the optical response with astandard response parameter; wherein the sample is immobilized in or ona solid or semi-solid matrix that is a membrane, an electrophoretic gel,a silicon chip, a microwell plate, or a microfluidic chip.
 11. Themethod according to claim 9, wherein the sample comprises cells; and/orfurther comprising tracing a temporal or spatial location of the opticalresponse with the sample.
 12. The method according to claim 9, whereinfor said compound S_(c) is an amino acid, a peptide, a protein, apolysaccharide, a nucleotide, a nucleoside, an oligonucleotide, anucleic acid polymer, an ion-complexing moiety, a lipid, or anon-biological organic polymer or polymeric microparticle, wherein S_(c)is optionally bound to one or more fluorophores that are the same ordifferent.
 13. The method according to claim 9, wherein for saidcompound, S_(c) is an ion-complexing moiety that is a BAPTA or an APTRA.